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Zinc depletion-induced apoptosis is associated with altered microRNA expression in human breast cancer… Bakker, Melinda 2013

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     Zinc depletion-induced apoptosis is associated with altered microRNA expression in  human breast cancer MDA-MB-231 cells    by  MELINDA BAKKER     A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE AND POSTDOCTROAL STUDIES  (HUMAN NUTRITION)         THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2013   ? Melinda Bakker, 2013   ii Abstract  Zinc is an essential trace element required for many physiological functions, including growth. At the cellular level, zinc is required for structural and catalytic roles in thousands of proteins, and adequate labile zinc is an important determinant of cellular viability. However, abnormal zinc accumulation in breast tissue is associated with breast cancer, suggesting that zinc status plays a role in breast cancer pathogenesis. Chelation-induced depletion of labile intracellular zinc promotes apoptosis, or programmed cell death, in multiple breast cancer cell lines. The mechanisms whereby zinc regulates apoptosis remain unclear. In particular, little is known about the role of microRNAs (miRs), a novel class of short non-coding RNA, involved in the regulation of gene expression. Zinc status can influence miR expression, and possibly the processing and stability of miRs. The hypothesis of my thesis research is that miRs are involved in zinc depletion-induced apoptosis in human breast cancer cells. The overall objective of this study was to determine the involvement of miRs in zinc depletion-induced apoptosis in breast cancer MDA-MB-231 cells. Zinc depletion for 24, 48 and 72 h induced apoptosis in 4.5, 24.4 and 28.0 % of the cells, respectively, indicating a time-dependent increase in zinc depletion-induced apoptosis. Expression of 8, 90, and 94 miRs were significantly altered during the early stages of zinc depletion-induced apoptosis, at 3, 12, and 24 h of zinc depletion, respectively. Overall, expression of 285 unique miRs was significantly affected by zinc depletion, duration of zinc depletion, and their interactions. qRT-PCR analysis confirmed that zinc depletion resulted in an increased abundance of miR-132-3p, miR-1246, miR-1273, miR-4484 and miR-4787-5p and a decreased abundance of miR-4521 in a time-dependent manner. MiR-132-3p and miR- iii 1246 have previously been shown to play a role in mediating apoptosis in prostate cancer PC-3 and lung cancer A549 cells, respectively. In conclusion, abundance of numerous miRs was altered during the early stages of zinc depletion-induced apoptosis, indicating possible involvement of these miRs in mediating zinc depletion-induced apoptosis. The role and targets of these miRs in zinc depletion-induced apoptosis requires validation in further research.  iv Preface This dissertation presents ?the ?findings ?of ?my ?master?s research study and was prepared in accordance with the requirements of the University of British Columbia Faculty of Graduate Studies. I designed the experiments together with Dr. Zhaoming Xu, and was responsible for performing all experiments and interpreting of the results under the supervision of Dr. Xu, with the exception of the microarray profiling experiment. LC Sciences performed the microarray profiling, as well as assisted with subsequent data analyses.   v Table of Contents  Abstract ................................................................................................................................... ii Preface .................................................................................................................................... iv Table of Contents .................................................................................................................... v List of Tables ......................................................................................................................... vii List of Figures ...................................................................................................................... viii Acknowledgements ................................................................................................................ xi Introduction ............................................................................................................................ 1 Chapter 1: Literature Review, Hypothesis, and Objectives ............................................... 3 1.1. Zinc Overview ............................................................................................................... 3 1.1.1. Introduction............................................................................................................. 3 1.1.2. Food sources ........................................................................................................... 5 1.1.3. Absorption, transport & excretion .......................................................................... 5 1.1.4. Cellular zinc homeostasis ....................................................................................... 8 1.2. Zinc and Breast Cancer ................................................................................................ 10 1.3. Zinc Depletion-Induced Apoptosis .............................................................................. 15 1.3.1. Introduction to apoptosis ...................................................................................... 15 1.3.2. In vivo research ..................................................................................................... 19 1.3.3. In vitro research .................................................................................................... 20 1.3.4. Mechanisms of zinc depletion-induced apoptosis ................................................ 23 1.4. MicroRNAs: Regulators of Apoptosis ........................................................................ 29 1.4.1. Introduction to microRNA .................................................................................... 29 1.4.2. MiR biogenesis ..................................................................................................... 30 1.4.3. MiR function ......................................................................................................... 31 1.4.4. MiR stability ......................................................................................................... 32 1.4.5. MiR expression in breast cancer ........................................................................... 33 1.4.6. MiRs & apoptosis in breast cancer ....................................................................... 36 1.5. Zinc and MiR Expression ............................................................................................ 40 1.6. Hypothesis ................................................................................................................... 44 1.7. Overall Objective and Specific Aims .......................................................................... 44 Chapter 2: Zinc depletion-induced apoptosis is associated with altered microRNA expression in human breast cancer MDA-MB-231 cells ................................................... 45 2.1. Materials and Methods ................................................................................................ 45 2.1.1. Cell culture system ............................................................................................... 45 2.1.2. Depletion of intracellular zinc .............................................................................. 45 2.1.3. Apoptosis assay .................................................................................................... 46 2.1.4. Total RNA isolation .............................................................................................. 47 2.1.5. MiR microarray assay ........................................................................................... 49 2.1.6. MiR heat maps ...................................................................................................... 50 2.1.7. qRT-PCR miR assay ............................................................................................. 50 2.1.8. Statistics ................................................................................................................ 52 2.2. Results ......................................................................................................................... 54 2.2.1. Zinc depletion-induced apoptosis ......................................................................... 54 2.2.2. Zinc depletion altered miR expression ................................................................. 55  vi 2.2.3. Zinc depletion altered abundance of miR-132-3p, miR-1246, miR-1273g-3p, miR-4484, miR-4521 and miR-4787-5p ........................................................................ 57 2.3. Discussion .................................................................................................................... 60 Chapter 3: Conclusions, Limitations, and Future Directions .......................................... 81 3.1. Conclusions ................................................................................................................. 81 3.2. Limitations ................................................................................................................... 82 3.3. Future Directions ......................................................................................................... 84 References .............................................................................................................................. 87 Appendices .......................................................................................................................... 106  vii List of Tables  Table 2.1: Differential expression of miR-182-5p induced by 3-h TPEN treatment. ............ 68 Table 2.2: Differential expression of miRs induced by 12-h TPEN treatment. ..................... 69 Table 2.3: Differential expression of miRs induced by 24-h TPEN treatment. ..................... 70 Table A.1: MiR expression in control and TPEN-treated MDA-MB-231 cells. .................. 107    viii List of Figures  Figure 1.1: Key elements of the intrinsic mitochondrial apoptotic pathway. ......................... 42 Figure 1.2: General pathway for microRNA (miR /miRNA) biogenesis and function. ......... 43 Figure 2.1: Zinc depletion-induced apoptosis in MDA-MB-231 cells. .................................. 71 Figure 2.2: 3-h TPEN treatment altered expression of several miRs. .................................... 72 Figure 2.3: 12-h TPEN treatment altered expression of many miRs. ..................................... 73 Figure 2.4: 24-h TPEN treatment altered expression of many miRs. ..................................... 74 Figure 2.5: Zinc depletion promoted a time-dependent increase in hsa-miR-132-3p expression. ...................................................................................................................... 75 Figure 2.6: Zinc depletion increased expression of hsa-miR-1246 after 12 and 24 h TPEN treatment. ........................................................................................................................ 76 Figure 2.7: Zinc depletion-induced time-dependent upregulation of hsa-miR-4484. ............ 77 Figure 2.8: Zinc depletion increased hsa-miR-4787-5p expression at 3 and 12 h. ................ 78 Figure 2.9: Zinc depletion promoted expression of a-miR-1273g-3p in a time-dependent manner. ........................................................................................................................... 79 Figure 2.10: Zinc depletion inhibited has-miR-4521 expression in a time-dependent manner. ........................................................................................................................................ 80 Figure A.1: Hsa-miR-16-5p expression measured by microarray (z-scores of log transformed signal intensities). ......................................................................................................... 122 Figure A.2: Hsa-miR-16-5p expression during qRT-PCR experiment 1. ............................ 123 Figure A.3: Hsa-miR-16-5p expression during qRT-PCR experiment 2. ............................ 124 Figure A.4: Hsa-miR-16-5p expression during qRT-PCR experiment 3. ............................ 125 Figure A.5: Hsa-miR-16-5p expression during qRT-PCR experiment 4. ............................ 126 Figure A.6: Baseline DNdA fragmentation in MDA-MB-231 cells at 0 h. ......................... 127 Figure A.7: DNA fragmentation in MDA-MB-231 cells treated with TPEN at 3 h. ........... 128 Figure A.8: DNA fragmentation in MDA-MB-231 cells treated with TPEN at 6 h. ........... 129 Figure A.9: DNA fragmentation in MDA-MB-231 cells treated with TPEN at 12 h. ......... 130 Figure A.10: DNA fragmentation in MDA-MB-231 cells treated with TPEN at 24 h. ....... 131 Figure A.11: 48-h TPEN-induced DNA fragmentation in MDA-MB-231 cells. ................. 132 Figure A.12: 72-h TPEN-induced DNA fragmentation in MDA-MB-231 cells. ................. 133   ix List of Abbreviations AIF Apoptosis inducing factor ANOVA Analysis of variance Ago Argonaute BAD  BCL-2-associated death promoter protein BAK1 BCL-2 antagonist killer 1 BAX  BCL-2 associated X protein BCL-2 B-cell lymphoma 2 BHK British hamster kidney BT-IC Breast tumor-initiating cells DCCR8 DiGeorge syndrome critical region 8 DCIS Ductal carcinoma in situ DIABLO Direct IAP binding protein with low pI DNAse Deoxyribonuclease DMEM  Dulbecco?s ?modified ?eagle?s ?medium DMSO  Dimethyl sulfoxide DTPA Diethylenetriaminepentacetic acid dNTP  Deoxyribonucleotide triphosphates dTTP  Deoxythymidine triphosphate DYRK1A  Dual specificity tyrosine-phosphorylation-regulated kinase 1A EDTA Ethylenediaminetetraacetic acid ER+/-  Estrogen receptor-positive/-negative FBS Fetal bovine serum FOXO Forkhead box O HEK Human embryonic kidney HER2 Human epidermal growth factor receptor type II H2O2 Hydrogen-peroxide Hsa Human HSD  Honestly Significant Difference IAPs Inhibitor of apoptosis proteins ICAD Inhibitor of caspase-activated deoxyribonuclease LIPZ  Labile intracellular pool of zinc MiR MicroRNA MMP  Mitochondrial membrane potential MRE Metal response elements MT  Metallothionien MTF-1 Metal-responsive-element binding transcription factor-1 MNU N-methyl-N-nitrosourea NAC N-Acetyl-L-Cysteine NF Nuclear factor NOS Nitric oxide synthase OMM Outer mitochondrial membrane PARP Poly ADP ribose polymerase PACT  Protein activator of PKR PBL Peripheral blood T lymphocytes  x PBS  Phosphate buffered saline PI  Propidium iodide PR Progesterone receptor Pre Precursor PrE Primary prostatic epithelial Pri Primary PTP Permeability transition pore qRT-PCR  quantitative reverse transcription polymerase chain reaction RISC RNA-induced silencing complex RNAse  Ribonuclease SD Standard deviation SirT  Silent information regulator Sp Specificity protein SSPE Saline-sodium phosphate-EDTA TNF  Tumor necrosis factor TPEN  N,N,N?,N?-tetrakis(2-pyridylmethyl)ethylenediamine TRBP Trans-activating response RNA-binding protein UTR  Untranslated region VSMCs Vascular smooth muscles cells XIAP X-linked inhibitor of apoptosis protein Zip  ZRT, IRT-like protein ZnT  Zinc transporter  xi Acknowledgements I am grateful to the many people who helped make this research possible. First of all, I would like to acknowledge and thank my supervisor Zhaoming Xu for numerous hours spent guiding and assisting me with this research project. I would also like to thank my supervisory committee: Christine Scaman, Vivien Measday and Amandio Vieira for their insightful advice and feedback during the course of my research studies. I would also like to thank my labmates both past and present for their assistance and support, especially Deanna Ibbitson who really encouraged me during our many coffee shop visits. I would like to offer a huge thank you to my husband Patrick for supporting me through the inevitable ups and downs ?of ?my ?studies. ?I ?can?t ?even ?describe ?all ?of ?the ?many ?ways ?you ?were ?there ?for ?me ?? not least of all visiting me when I was stuck in the lab for long hours, patiently listing to technobabble about my research, challenging me to grow as a person, providing endless encouragement and loving me unconditionally. I am thankful for the continued love and support of my family, especially my mom and dad, and my siblings Charlene, Brent and Katie. Thank you also mom and dad Bakker for your love and encouragement. A big thank you to the rest of the extended Ouwerkerk and Bakker family for your support, especially to Jonathan Bakker for being a sounding board on my research design and statistical analysis. I am grateful for the support I received from friends, with special thanks to Nick and Renee Reeves for the their infectiously positive attitude and frequent encouragement. I am thankful to have had the opportunity to pursue this research and for the many people who supported me throughout!   1 Introduction Zinc is an essential nutrient, required for a broad range of physiological functions in humans, including growth, reproduction, normal neurological function and immunity1. However, zinc accumulation has been associated with breast cancer2. Furthermore, elevated expression of some zinc transporters and the zinc storage protein metallothionein are implicated in breast cancer and have been found to regulate some cancer traits, including resistance to apoptosis3?10.  Zinc is known to regulate apoptosis and zinc depletion has been shown to induce apoptosis both in vivo and in vitro11,12. In multiple breast cancer cell lines, depletion of intracellular zinc promoted apoptosis through the intrinsic mitochondrial apoptotic pathway, involving release of cytochrome c from the mitochondria and activation of the caspase cascade13,14, though the exact mechanisms involved are not fully understood. Currently, little is known about the role of microRNAs (miRs) in mediating zinc depletion-induced apoptosis. MiRs are small non-coding RNAs that regulate gene expression at the post-transcriptional level15. MiRs regulate numerous signaling pathways, including key apoptotic pathways in cancer16,17. Treatments targeting expression of miRs have been shown to influence cancer pathogenesis18.  Only a few studies to date have investigated the effects of zinc status on miR expression, finding that zinc regulates miR expression both in vivo19,20 and in vitro21,22, and may influence the processing and stability of miRs21. Therefore, miRs might play a role in zinc depletion-induced apoptosis. This research is intended to investigate the possible  2 involvement of miRs in zinc depletion-induced apoptosis using human breast cancer MDA-MB-231 cells as a model. An improved understanding of the mechanisms involved in zinc-mediated regulation of apoptosis may contribute to the development of new treatment strategies against breast cancer.  3 Chapter 1: Literature Review, Hypothesis, and Objectives 1.1. Zinc Overview 1.1.1. Introduction Zinc is an essential trace element and the second most abundant trace element in humans after iron, with 2-3 grams of total zinc in an adult1. Zinc is present in all tissues, with 90% of the body zinc in the muscle and bones23. Zinc is present only as a divalent cation (Zn2+) in living organisms. Zinc exerts a wide range of physiological functions.  One of the most profound functions of zinc is its essential role in growth - zinc deficiency impairs growth in every living organism studied to date24. Zinc has many other physiological functions, including reproduction, brain development, immunity, taste and vision, etc.1. Zinc deficiency causes many adverse health effects; early symptoms of zinc deficiency are growth retardation, skin lesions, and immune dysfunction1.  Additionally, hair loss and poor wound healing also occur with zinc deficiency1. Severe zinc deficiency can cause hypogonadism in male adolescents, failed pregnancies, diarrhea, infections and neurological disorders1. In humans, acrodermatitis enteropathica, a genetic disorder resulting in decreased absorption of zinc, results in the typical broad range signs of zinc deficiency including impaired growth, dermatitis, diarrhea, immune dysfunction and sometimes neuropsychological disorders25.  At the cellular level, zinc plays a role in numerous cellular signaling pathways, including those involved in cell proliferation, differentiation and apoptosis26. Zinc deficiency  4 has been shown to impair DNA synthesis27, reduce cell viability, and induce apoptotic cell death14.  Zinc is present in thousands of proteins, including many enzymes, which are known as zinc metalloenzymes, and transcription factors. Collectively, zinc metalloenzymes and zinc-containing transcription factors are known as zinc metalloproteins.  Zinc metalloproteins have wide ranging functions, including their involvement in metabolism of proteins, carbohydrates, nucleic acids and lipids, as well as regulation of gene expression24. Using a bioinformatics approach, it is predicted that 10 % of the human proteome is made up of zinc-containing proteins, of which 40% are transcription factors, while the remainder is made up mostly of zinc metalloenzymes28. Zinc-containing transcription factors are zinc-finger proteins, in which zinc is bound to amino acids, most commonly 4 cysteine (Cys4) residues, or two cysteine residues followed by two histidine residues (Cys2His2)28. A major role for the zinc-finger structure is to interact with DNA base pairs, which is essential for the binding of transcription factors to their target nucleic acid sequence29. In zinc metalloenzymes, zinc plays both structural and catalytic functions. Zinc metalloenzymes belong to every enzyme classification, demonstrating a wide ranging role for zinc in enzyme function24. Another recently identified, but less well understood role of zinc, is that free zinc ion (Zn2+) has been shown to act as a second messenger in some cellular signaling pathways30.     5 1.1.2. Food sources The zinc content of foods is associated with its protein content. Thus, meats are generally good sources of zinc. Shellfish such as oysters are particularly rich in zinc (33.2-182.0 mg Zn/100g cooked oyster) and red meat is also a good source of zinc (e.g. cooked beef: 5.3 ? 11.5 mg Zn/100g)31. White meats and finfish contain lower amounts of zinc (e.g. cooked turkey: 1.1 -3.6 mg Zn/100 g; chicken: 1.1 ? 2.9 mg Zn/100g; wild atlantic salmon: 0.8 mg Zn/100 g)31. Milk (1.0-1.1 mg Zn/250 mL cup) and dairy products also provide a source of zinc 31. Zinc is also found in whole grains (e.g. wheat germ: 8.0 mg Zn/100g) and legumes (ex. cooked lentils: 1.3 mg Zn/100g; cooked peas: 1.5 mg Zn/100g)31. While zinc is found in protein rich plant foods, the bioavailability of zinc in plant foods is generally lower due to the presence of inhibitors of absorption (e.g. phytic acid, oxalic acid and polyphenols, etc.)1. Thus, fruits and vegetables are generally poor sources of zinc31. 1.1.3. Absorption, transport & excretion  During digestion, dietary zinc is hydrolyzed from amino acids and nucleic acids32. The solubility of zinc influences absorption. Plant ligands such as phytate (phosphorous storage form in plants) can bind to zinc forming insoluble complexes, making it unavailable for absorption33,34. Animal protein generally offers a higher bioavailability of zinc; furthermore, it has been shown to improve absorption of zinc from plant sources when they are ingested at the same time35,36.   6 The gastrointestinal system is mainly responsible for regulating systemic zinc homeostasis through absorption of dietary zinc and excretion of endogenous zinc25,37,38. Zinc is absorbed in throughout the small intestine, with the jejunum being the main site of absorption39. Humans consuming diets low in zinc generally absorb zinc with a higher efficiency and are adapted to increase zinc absorption over time40. However, high phytate consumption prevents positive adaptations in zinc absorption during low zinc consumption. The efficiency of zinc absorption is generally inversely related to dietary zinc intakes; however, the quantity of dietary zinc absorbed increases with higher dietary zinc intakes.   Only recently have the molecular mechanisms of zinc absorption began to be elucidated, due to increased understanding of the role of zinc transporters in enterocytes, or intestinal absorptive cells. In humans, there are two families of zinc transporters: the ZnT or SLC30A family and Zip (Zrt-, Irt-like proteins) or SLC39A family. To date, 10 ZnT and 14 Zip transporters have been identified in humans41. In 2002, it was discovered that mutation in Zip4 (Zrt-, Irt-like protein 4), a novel zinc transporter, was responsible for acrodermatitis enteropathica42,43. In mice, dietary zinc deficiency caused upregulation of Zip4 expression and promoted its localization to the apical plasma membrane of enterocytes44,45. Additionally, another zinc transporter ZnT5B was found to be involved in zinc uptake in human Caco-2 cells46.  In addition to dietary zinc absorption, the small intestine also reabsorbs zinc from endogenous secretions. Zinc is found in high levels in salivary, gastric, pancreatic and biliary secretions47. Reabsorption of endogenous zinc appears to be crucial to reaching positive zinc balance during low zinc intakes, more so than absorption of dietary zinc. In a human study,  7 when male subjects consumed a low zinc diet (4.1 mg Zn/day) for 6 months, the fractional zinc absorption rate was increased, but the total dietary zinc absorption still decreased compared to the baseline48. In compensation, fecal losses of endogenous zinc were reduced, resulting in a net crude positive zinc balance after 6 months. In another study, zinc homeostasis in young Chinese women from either a rural farming area with low zinc intakes (5.2 mg Zn/day) or an urban area with higher intakes (8.1 mg Zn/d) were compared49. There was no difference in fractional zinc absorption between these two groups of women, indicating that the rural women absorbed significantly lower zinc in proportion to the low dietary intake. However losses of endogenous zinc were decreased in the rural women, offsetting the lower amount of dietary zinc absorbed so that the total amount of zinc absorbed was similar between the two groups. Upon entering the enterocyte, zinc may be utilized and/or stored locally. Absorbed zinc is also transported across the basolateral membrane into the portal vein. The ZnT1 transporter is thought to control release of zinc from enterocytes into the portal vein due to its location on the basolateral membrane50,51 and role in zinc efflux from cultured cells52. About 60% of zinc in general circulation is bound to albumin for transport32. Zinc may also be bound to transferrin, ?-2 macroglobulin, histidine and cysteine for transport32. The serum zinc makes up only 0.1% of the total body zinc, but it supplies tissues with the necessary zinc and undergoes quick turnover53. Plasma zinc is redistributed to tissues during a variety of conditions (e.g. inflammation, infection, trauma, stress and the postprandial period all decrease plasma zinc)54.  8 The gastrointestinal tract is the primary location for loss of zinc from the body due to fecal excretion55. Zinc may also be lost in smaller amounts through urine, sweat, shed skin, nails, hair, menstruation and semen37. In summary, systemic zinc homeostasis is maintained primarily at the gastrointestinal tract through regulation of dietary zinc absorption and endogenous secretion of zinc. Fractional absorption of dietary zinc tends to be inversely related to dietary zinc intakes. Reductions in endogenous zinc losses are a critical factor in conserving zinc status during low dietary intakes. 1.1.4. Cellular zinc homeostasis The total cellular zinc concentration is typically maintained around a few hundred micromolar ?(e.g. ?264 ??M ?in ?human ?colon ?cancer ?HT-29)56. While zinc is essential for normal cellular function, it is also toxic in excess. Therefore, total cellular zinc content and its intracellular distribution must be tightly regulated, balancing its requirement for numerous structural and enzymatic roles while preventing potential toxicity. The majority of cellular zinc is bound to metalloproteins with a high binding affinity so that free zinc (Zn2+) concentration is typically maintained at extremely low levels (5 - <1000 pM in various cells and tissues), which is about 6-7 orders of magnitude lower than the total cellular zinc concentration57. In order to maintain zinc homeostasis, complex regulatory systems are involved, particularly at the level of zinc transport, storage, and sensing.  Sequestration of zinc into subcellular organelles and controlled release is important for homeostatic control of free Zn2+ level58. Typically, about half the intracellular zinc is  9 located in the cytosol as well as subcellular organelles including zincosomes, a vesicular structure with highly concentrated levels of zinc24. An additional 30 to 40 % of the intracellular zinc is located in the nucleus24. The remainder of the zinc is associated with the cell membranes24.  Zinc transporters regulate the movement of extracellular zinc into the cell as well as controlling the intracellular zinc levels, keeping free intracellular zinc (Zn2+) at low levels. ZnT transporters are generally responsible for zinc efflux from the cytosol, sequestering zinc into subcellular organelles or transporting zinc to the extracellular space59. However, ZnT5B is an exception to this rule of thumb, acting as a bidirectional zinc transporter46. Zip transporters have been shown to increase cytosolic zinc, by extracellular zinc uptake and release from intracellular vesicles59. The expression of ZnTs and Zips is tissue/cell type specific and regulation of their expression is an area of ongoing research. Another important family of proteins involved in zinc homeostasis is metallothioneins (MTs), metal-binding proteins that mediate zinc storage. MTs have high capacity for zinc due to the large number of cysteine residues (1/3 amino acids) with sulphur ligands that bind to zinc57. Four major isoforms of mammalian metallothionein have been identified (MT1-4), based on similarities in their amino acid sequences. In humans, there are 11 known functional MT-1 genes (MT1-A, -B, -E, -F, -G, -H, -I, -J, -K, -L and ?X) and one gene for the other three isoforms (MT-2A, MT-3 and MT-4)7. MT-1 and MT-2 are widely expressed across different tissues, while MT-3 is expressed in the brain and MT-4 is expressed in squamous epithelia60. Each MT molecule binds up to 7 zinc ions, exhibiting at least three different levels of binding affinities from nanomolar to picomolar of zinc61. When  10 the level of available zinc increases, metallothionein synthesis is induced to buffer the zinc62. On the other hand, MT may function as a zinc donor when available zinc decreases62. The metal-responsive-element binding transcription factor-1 (MTF-1) is an important zinc sensor. It responds to increases in intracellular zinc by activation of genes involved in zinc storage and zinc transport, thus playing an important role in homeostatic control of cellular zinc levels63,64. Free zinc (Zn2+) binds to MTF-1 causing it to rapidly translocate from the cytosol to the nucleus, where it binds to the metal response elements (MRE) in the promoter region of the MT gene65 and activates the transcription of metallothionein66?68. MTF-1 has also been shown to mediate zinc induced expression of ZnT1 in mice69.  In summary, zinc is an essential trace element and its absorption, transport and excretion are tightly regulated in order to provide the body with adequate zinc when possible. At the cellular level, the movement of free zinc ions is also finely regulated in order to balance requirements for cellular functions against potential toxicity. When zinc homeostasis is altered due to changes in expression of zinc transporters and metallothionein, it may lead to the development and progression of disease, including breast cancer, which will be discussed more in the following section70,71. 1.2. Zinc and Breast Cancer Abnormal zinc status is implicated in the development of some diseases, including certain types of cancer2,72; however, the role of zinc in tumor development and growth is poorly understood. In studies with rats, zinc was found to accumulate in mammary tumors  11 induced with the carcinogen N-methyl-N-nitrosourea (MNU) at significantly higher levels compared to healthy mammary tissue (6-19 times)73.  Zinc is an important mineral for tumor growth, because it is required for cell proliferation27 and zinc deficiency suppresses cell proliferation and induces apoptosis27,74. Zinc deficiency has been shown to inhibit the growth of a wide range of tumors in animals75?79. When rats were fed a zinc-deficient diet following the establishment of implanted mammary adenocarcinomas, tumor growth was suppressed80. This study also found that there was a positive correlation between the tumor zinc concentration and the percent viable tumor tissue. However, the zinc concentration in the tumors did not vary significantly between the rats fed a zinc-deficient diet (<4 mg Zn/kg diet) and the rats fed one of the three control diets, depending on the experiment (35-50 Zn/kg diet). In another study, zinc deficiency also protected against the development of MNU-induced mammary tumors in rats81.  Interestingly, zinc was found to accumulate in rat mammary tumors regardless of whether or not the rats consumed a zinc-deficient (3 mg Zn/kg diet) or zinc-adequate (31 mg Zn/kg diet) diet, despite the ten-fold difference in zinc dietary content and there was no significant difference in the zinc content of the tumors between the rats fed the different diets73. These animal studies suggest that zinc deficiency may protect against cancer development and growth, possibly due to a requirement for a higher level of zinc in cancer growth.  While zinc is important for tumor growth, high levels of dietary zinc intake have not been found to promote mammary cancer growth. In nude mice xenografted with human MCF-7 breast cancer cells, the primary tumor growth rate did not differ between the rats fed  12 a zinc supplemented diet (180 mg Zn/kg diet) and the rats fed a zinc adequate diet (30 mg Zn/kg diet)82.  Similar to animal studies, multiple human clinical studies also indicate that zinc accumulates in malignant breast tissue83?95. Comparisons of cancerous breast tissue to adjacent healthy tissue from the same patient indicate that zinc concentration is elevated ranging from a 1.4 ? 4.4 fold increase (median concentration)83,84,86?93. Recent advances in measuring trace elements using x-ray fluorescence have permitted the assessment of trace elements distribution in breast tissue with higher spatial resolution. Malignant breast tissue samples display accumulation of zinc in areas of cancer cell clusters, with an 87% increase in zinc concentration94. Non-paired comparisons of the zinc level in breast cancer tissues to breast tissue samples from healthy women also indicates an elevated zinc level in breast cancer tissues, with an increase ranging from 2.6 to 5.2 fold (median concentration)86,87,91,93,95. Zinc appears to be more likely to accumulate in estrogen receptor positive (ER+) breast cancer cells, as ER+ breast tumors contain 80% more zinc than ER- breast tumors96. Some studies have found that zinc was elevated in benign breast disease compared to healthy breast tissue in different women93,95, but another study found that there was no difference in tissue zinc concentrations between paired samples of benign breast tumors and normal breast tissue from the same patients89. One study investigated the association between zinc levels in women with benign breast disease and subsequent risk of breast cancer, finding that higher zinc levels in breast tissue was associated with a modest increase in the risk of developing breast cancer later in life97.  13 The mechanisms of elevated zinc in malignant breast tissue may be due to alterations in expression of zinc transporters as well as metallothioneins. Zinc accumulation in NMU-induced rat mammary tumors was associated with alterations in zinc homeostasis, specifically decreased expression of the ZnT1 transporter, involved in zinc efflux, and increased expression of metallothionein, the zinc storage protein98. In human breast cancer, both in vivo and in vitro research indicate abnormal expression of multiple zinc transporters including Zip6, Zip7, Zip10 and ZnT23?6,99,100, as well as upregulation of metallothionein7; and these findings will be discussed in more detail in the following sections.  Zip6 (LIV-1), is a zinc importer that is initially expressed as a pro-protein at the endoplasmic reticulum , then cleaved on the N-terminus prior to localization at the plasma membrane101. Expression of Zip6 increases in response to estrogen and it has been found to be frequently upregulated in estrogen-receptor positive breast cancer99,100,102. Increased expression of Zip6 has been associated with metastasis of breast cancer to the lymph nodes3. On the other hand, high expression of Zip6 protein has also been associated with increased survival in patients with invasive ductal carcinomas103. However, the antibody used in this study targeted the Zip6 pro-protein form located on the endoplasmic reticulum, not the active plasma membrane protein103,104. Research studies in vitro provide conflicting evidence. One study found that Zip6 upregulation in MCF-7 cells promoted epithelial-mesenchymal transition, a critical step in tumor metastasis104. In contrast, another study found that Zip-6-attenuation decreased apoptosis, increased tumor colony formation, and reduced E-cadherin expression (an epithelial marker) in T47D breast cancer cells105.   14 Zip7 is located on the endoplasmic reticulum, and is thought to control release of zinc from ER stores to the cytoplasm106. Increased expression of Zip7 was associated with acquired tamoxifen resistance in MCF-7 breast cancer cells4. In this study, Zip7 upregulation was found to increase intracellular zinc and to activate an oncogenic growth factor signaling pathway. Furthermore, inhibition of Zip7 reduced intracellular zinc and inhibited breast cancer cell migration.  Elevated expression of Zip10, a zinc importer, was associated with breast cancer lymph node metastasis5. Further research revealed that Zip10 was associated with more aggressive breast cancer cell lines (i.e. MDA-MB-231), than non-invasive breast cancer cell lines (i.e. MCF-7). Zip10 mediated zinc import was essential to the invasive behavior of breast cancer cells, as depletion of either Zip10 or intracellular zinc inhibited migration of metastatic breast cancer cell lines. The zinc transporter ZnT2 is involved in transporting zinc into vesicles and has been found to protect zinc-sensitive baby hamster kidney (BHK) cells from excess zinc accumulation107. ZnT2 may also play a role in protecting breast cancer cells from the cytotoxic effects of elevated cytoplasmic zinc. Increased expression of ZnT2 was observed in MT-null T47D breast cancer cells compared to non-malignant MT+ MCF-10A breast cells6. Additionally, inhibition of ZnT2 increased cytoplasmic zinc and resulted in increased apoptosis and reduced tumor formation.   Breast cancer has also been associated with altered zinc storage, as evidenced by changes in MT expression. The majority of studies have found that increased expression of MT-1 and -2 were associated with tumor grade, tumor stage and poor survival in breast  15 cancer7. However, some studies failed to find an association between MT-1 and -2 and survival of breast cancer patients7. The MT-2A isoform is more highly expressed in invasive breast cancer cell lines and may play a role in breast cancer progression. Overexpression of MT-2A in human MCF-7 breast cancer cells increased cellular proliferation, while silencing MT-2A caused growth arrest and apoptosis8. In another study, inhibition of MT-2A inhibited cell cycle progression, but only caused a marginal increase in apoptosis in MCF-7 cells9. Silencing of MT-2A expression inhibited migration and invasion of human MDA-MB-231 breast cancer cells10. MT transcription is regulated by MTF-1, which was found to be elevated in human breast cancer tissues compared to normal adjacent tissue108. In summary, zinc appears to be accumulated in breast cancer tissues.  The significance of this apparent zinc accumulation in breast cancer development and progression remains unclear. Accumulation of zinc in breast cancer cells appears to result from an altered zinc homeostasis, particularly the expression of zinc transporters (i.e. Zip6, Zip10) and metallothionein. Therefore, zinc appears to play an important role in breast cancer pathogenesis.  1.3. Zinc Depletion-Induced Apoptosis 1.3.1. Introduction to apoptosis Apoptosis or gene-directed cell death is the major type of cell death in the body for elimination of unneeded, mutant or moderately damaged cells109. While apoptosis is needed during development and tissue remodeling, defects in apoptosis contribute to pathological  16 conditions such as cancer110. It is an energy-dependent process involving complex coordination of signalling pathways and subsequent morphological changes including cell shrinkage, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies.  Apoptosis is a highly regulated process that can be initiated by three different cellular signalling pathways: the extrinsic death receptor pathway, the intrinsic mitochondrial pathway and the intrinsic endoplasmic reticulum pathway.  The death receptor pathway is activated by the binding of an extracellular apoptotic ligand [i.e. Fas, tumor necrosis factor (TNF)-? ?and ?TNF-related apoptosis-inducing ligand receptors] to its receptor in the plasma membrane111. The intrinsic mitochondrial pathway may be initiated by intracellular stresses such as toxins, hypoxia, oxidative stress, UV and gamma irradiation, but the signalling pathways involved are still unclear111. The intrinsic endoplasmic reticulum pathway is not as well studied, but it involves accumulation of unfolded protein and impaired protein synthesis following ER damage from hypoxia, free radicals or glucose starvation112. For the purpose of this thesis, the focus is on the intrinsic mitochondrial pathway.  Upon activation of the intrinsic mitochondrial pathway, the outer mitochondrial membrane (OMM) becomes permeable (Figure ). The B-cell lymphoma 2 (BCL-2) protein family and the permeability transition pore (PTP) regulate permeabilization of the OMM. The BCL-2 family contains the conserved BCL-2 homology domains and is made up of both anti-apoptotic proteins [i.e. BCL-2] and pro-apoptotic proteins [i.e. BCL-2 associated X protein (BAX)]. The relative abundance of pro- and anti-apoptotic proteins, localization to the mitochondria and conformational changes determines their ability to form channels in  17 OMM, resulting in permeabilization111. OMM permeabilization may also be achieved by opening of the PTP, an unselective mitochondrial channel, causing swelling of the mitochondrial matrix and rupture of the OMM111. There appears to be cross-talk between these two pathways as proteins in the Bcl-2 family have been shown to regulate PTP activity113. As a result of either pathway, increased OMM permeabilization permits release of proapoptotic proteins from the intermembrane space into the cytosol111,113.  One such protein, cytochrome c is typically located in the mitochondrial inter-membrane space in healthy cells, but is released into the cytosol during apoptosis114. Cytochrome c is a key activator of caspases, a family of cysteine proteases that mediate apoptosis. In the cytosol, cytochrome c binds to apoptotic protease activating factor (Apaf1), forming a multimeric apoptosome115. The apoptosome recruits and activates caspase-9115. Once activated caspase-9 cleaves the downstream executioner caspases such as caspase-3, which in turn cleave key substrates involved in apoptosis115.  Permeabilization of the OMM also causes the release the proapototic proteins Smac (second mitochondrial activator of caspases)/DIABLO (direct IAP binding protein with low pI) and HtrA2/Omi., which bind to inhibitor of apoptosis proteins (IAPs)116. IAPs bind to caspase-9 and caspase-3 to inhibit their activation and activity. Binding of Smac/DIABLO and HtrA2/Omi displaces caspases from IAPs, thus facilitating apoptosis116.  Additionally, there is some evidence that apoptotic stimuli release other pro-apoptotic proteins from the intermembrane space, which are involved in caspase-independent apoptotic pathways. For example, apoptosis inducing factor (AIF) may be released from the mitochondria and translocate to the nucleus, causing DNA fragmentation  18 and chromatin condensation resulting in apoptosis117. However, release of AIF is not a universal requirement for cell death and its role in mediating apoptosis varies in a cell type specific manner117.  Caspase mediated cleavage of hundreds of protein targets fosters apoptosis and is also thought to be responsible for most of the characteristic morphological changes observed during apoptosis118. Caspase-3 cleaves the inhibitor of caspase-activated DNAse (ICAD), activating CAD (caspase-activated-DNAse) and resulting in fragmentation of genomic DNA, a hallmark of apoptosis111. Caspase-3 mediated cleavage of DNA typically causes chromatin condensation118,119. Caspases also cleave many other key cellular components including cytoskeleton proteins and the nuclear scaffold (lamins), likely causing cellular shrinkage and membrane blebbing, as well as nuclear fragmentation118. Additionally, caspases target a number of cell adhesion proteins, which may play a role in cellular detachment from the extracellular matrix, however, the exact mechanisms involved in cellular detachment are still unclear118. In summary, apoptosis is an energy-dependent, highly controlled process for elimination of unwanted or damaged cells. Much of the signalling pathways implicated in apoptosis surround the regulation of caspase activation. In the intrinsic mitochondrial apoptotic pathway, permeabilization of the OMM facilitates the release of pro-apoptotic factors, such as cytochrome c, leading to caspase activation. Caspases play a key role in apoptosis by targeting hundreds of proteins to facilitate DNA fragmentation and controlled cellular breakdown.   19 Numerous studies have implicated zinc deficiency as a stimulus for apoptosis. The effects of zinc deficiency on apoptosis and the signalling pathways involved will be discussed in depth in the following sections. 1.3.2. In vivo research The apoptotic effects of zinc deficiency were first reported in 1977, when it shown that zinc deficient rats had increased apoptotic cells in their intestinal crypts120. Elevated apoptosis was also observed in chickens fed zinc-deficient diets in the tibial growth plate chrondrocytes121. More recently, marginal dietary zinc deficiency in rats was found to induce apoptosis in the vascular smooth muscles cells (VSMCs) of large arteries122.  One area of focus in understanding the role of zinc deficiency in apoptosis is the effects of zinc status on the immune system, since zinc deficiency causes lymphopenia and thymic atrophy, resulting in suppression of immune function12. In male rats fed a zinc free diet, increased apoptosis in the thymus was observed after just 1 week, which was attributed to the subsequent development of thymic atrophy after 4 weeks123. In mice fed a zinc-deficient diet, thymic atrophy was also associated with a marked increase in apoptosis in the thymus, specifically within the population of immature pre-T cells124.  Consumption of a zinc free diet has been shown to display time-dependent apoptotic effects in other tissues. In male rats fed a zinc free diet, increased apoptosis was also observed in the testes after 3 weeks, prior to the occurrence of testicular atrophy after 10 weeks123. With more prolonged zinc depletion, elevated apoptosis was observed in the kidney after 13 weeks and in the liver and skin after 34 weeks123.   20 Adequate zinc is required for development and zinc deficiency is teratogenic74. For example, consumption of a low zinc diet by pregnant rats, was associated with increased apoptosis in the embryos, especially within the neural crest cells125. Increased caspase-3 activity was associated with zinc deficiency-induced embryonic cell death at mid-gestation in rats126. Additionally, the effects of zinc deficiency on early embryonic development during implantation were investigated using a murine model127. In this model, blastocyst stage embryos taken from mouse dams were cultured in low-zinc, zinc-replete or control medium. Culturing in low zinc resulted in abnormal embryonic morphology, including smaller embryos, which was attributed to the increased apoptosis observed in the zinc-deficient embryos. In summary, zinc deficiency causes apoptosis in a wide variety of tissues. Zinc deficiency-induced apoptosis has been associated with impaired development, particularly during embryonic development. 1.3.3. In vitro research Zinc depletion-induced apoptosis has been demonstrated in numerous studies in vitro, and may be achieved by culturing cells in low or zinc-free medium, or alternatively by chelating zinc11. Apoptosis was induced by culturing human lymphoid (Raji) and myeloid (HL-60) cells in a zinc-free medium128 and by culturing human leukemia (HL-60, ?0.5 ??M129; Jurkat T-cells, ?1.5 ??M130) and neuroblastoma cells (IMR-32, ?1.5 ??M) ?in ?a ?low-zinc medium130. In addition, apoptosis can also be induced by using a chelator to deplete intracellular zinc, most commonly using N,N,N?,N?-tetrakis(2- 21 pyridylmethyl)ethylenediamine (TPEN).  TPEN is a membrane permeable heavy metal chelator with a higher affinity for zinc and a lower affinity for other divalent cations such as calcium and magnesium131. TPEN has been found to sequester zinc from the labile intracellular pool of zinc (LIPZ). Depletion of intracellular zinc using TPEN treatment (30 ?M) occurs within 20 min in HeLa cells132. Treatment of breast cancer cells (i.e. MCF-7, MDA-MB-468) with TPEN reduced the abundance labile zinc in a dose-dependent manner14. In MDA-MB-231 breast cancer cells, TPEN treatment reduced LIPZ in a dose- and time-dependent manner133. ?TPEN ?treatment ?at ?higher ?concentrations ?(10 ?and ?20 ??M) ?for ?8 h depleted the LIPZ to below detection in MDA-MB-231 cells133. Recently, it was discovered that TPEN also sequesters zinc from a large fraction of the zinc proteome. Treatment of LLC-PK1 pig ?kidney ?cells ?with ?25 ??M ?TPEN ?for ?30 ?min ?reduced ?the ?zinc ?bound proteome by 34% and Zn-MT by 50%134.  Chelation of intracellular zinc via TPEN (1 ? 100 ?M) ?induces ?apoptosis ?in ?many ?different human cell lines which include thymocytes135, lymphocytes136,137, malignant airway epithelial cells138, leukemia cells139, retinal pigment epithelial cells140, melanoma cells141, keratinocytes142, pancreatic cancer cells143, cervical cancer cells132 and breast cancer cells13,14,133. In breast cancer MDA-MB-468 and MCF-7 cells, chelation of either labile intracellular (via TPEN) or extracellular [via diethylenetriaminepentacetic acid (DTPA)] zinc induces apoptosis14. Previous research from our laboratory indicates that the human breast cancer cell lines MDA-MB-231, MCF-7 and T47D undergo apoptosis following TPEN-induced zinc depletion133. The level of apoptosis in these three breast cancer cell lines exceeded that observed in MCF-10A, a non-tumorigenic fibrocystic breast epithelial cell line.   22 The extent of the LIPZ depletion correlates well with the induction of apoptosis. In thymocytes from aged rats, which undergo spontaneous apoptosis, small decreases in the LIPZ (TPEN treatment) sharply increased the susceptibility to apoptosis, while elevating LIPZ (zinc plus an ionophore treatment) inhibited apoptosis144. TPEN-induced apoptosis in rat spleen cells was also associated with a decrease in the LIPZ144. In a human promyelocytic leukemia cell line (HL-60) grown in a low-zinc medium (5 ?M), loss of the LIPZ occurred prior to detection of mitochondrial membrane potential loss and apoptosis129. Furthermore, the LIPZ was significantly lower in cells identified as undergoing an early stage of apoptosis compared to healthy cells, regardless of the zinc concentration in the media (0.5, 25 & 50 ?M).  TPEN induced apoptosis may be prevented by concurrent addition of zinc, providing further evidence that TPEN-induced apoptosis is caused by zinc depletion. In MDA-MB-231 breast cancer cells, zinc treatment (10 ? 40 ??M), ?essentially ?prevented ?TPEN-induced apoptosis, as it was associated with a 99% reduction in DNA fragmentation13. Zinc supplementation also inhibited apoptosis in DTPA and TPEN treated MCF-7 and MDA-MB-468 cells14.  Zinc depletion may also increase susceptibility to apoptosis from toxins and other pro-apoptotic stimuli109. For example, TPEN-induced zinc depletion of porcine airway epithelial ?cells ?was ?found ?to ?synergistically ?increase ?tumor ?necrosis ?factor ?? ?and ?linoleic ?acid ?induced apoptosis145. Similarly, zinc deficiency also increased hydrogen-peroxide (H2O2)-induced caspase activation in respiratory airway cells138.   23 Some studies suggest that zinc supplementation may have anti-apoptotic effects109. Zinc supplementation inhibited H2O2-induced caspase activation in the model described above138. In human chronic lymphocytic leukemia cells, elevating the LIPZ via zinc supplementation in the presence of pyrithione, an ionophore, dramatically reduced the susceptibility to apoptosis induced by the toxin colchicine in a dose-dependent manner144. Increased labile zinc also inhibited apoptosis from a lipopolysaccharide endotoxin, as well as spontaneous apoptosis in sheep pulmonary artery endothelial cells146.  While zinc generally protects against apoptosis, its effects as an apoptotic regulator are cell type and dose-dependent. For example, in mouse thymocytes, zinc supplementation between 500- -induced apoptosis. In contrast, lower zinc supplementation between 80 ? 147.  In summary, zinc has been shown to play a critical role in the regulation of apoptosis. Zinc depletion may induce apoptosis and/or increase susceptibility to apoptotic stimuli. However, the effect of zinc on apoptosis is cell-type and dose-dependent.  1.3.4. Mechanisms of zinc depletion-induced apoptosis Zinc depletion-induced apoptosis is associated with alterations in apoptotic signalling pathways, specifically the intrinsic mitochondrial pathway, involving cytochrome-c release, and the caspase cascade. Studies of zinc depletion-induced apoptosis in breast cancer cells support the role of intracellular calcium influx, and breakdown of the X-linked inhibitor of apoptosis protein (XIAP) in the regulation of apoptosis. Additionally, oxidative stress is implicated in many studies of zinc depletion-induced cell death.  24 A number of studies have documented release of cytochrome c from the inner mitochondrial membrane into the cytosol following zinc depletion in peripheral blood T lymphocytes (PBL)137, breast cancer cells (i.e. MDA-MB-468, MCF-714, and MDA-MB-23113), neuronal148 and osteoblastic cells149, indicating that zinc depletion-induced apoptosis is associated with the intrinsic mitochondrial apoptotic pathway. The time-course of apoptosis was investigated in PBL cells, which showed that cytochrome c rapidly accumulated in the cytosol after just one hour of TPEN treatment (15 ?M), which was also associated with activation of caspase-3 and caspase-9137. After 2 h of TPEN treatment, the caspase-3 substrate poly ADP ribose polymerase (PARP) was cleaved. The release of cytochrome c is known to be an important factor in activating the caspase cascade115, and many studies support a role for altered expression of caspases in zinc depletion-induced apoptosis109. In rat embryos, zinc deficiency increased caspase-3 activity and apoptosis126. In multiple human malignant epithelial cells (colonic LIM1215, bronchial NCI-H292), TPEN treatment (25 ?M) was associated with rapid induction of caspase-3 (1-2 h), followed shortly by activation of caspase-6 (2 h in LIM1215 cells; 3-4 h in NCI-H292 cells)138,150. In breast cancer cells, depletion of intracellular (TPEN) and extracellular (DTPA) zinc for 48 h increased activity of caspase-9 and caspase-3 in MDA-MB-468 cells and activity of caspase-9 in MCF-7 cells (MCF-7 cells lack caspase-3) in a dose-dependent manner14. Furthermore, there was no change in caspase-8 activity following zinc depletion, indicating that the death receptor pathway was not involved in mediating apoptosis. Research from our laboratory also showed that zinc depletion-induced apoptosis was linked to early changes in caspase-9 and -3 activity (3-6 h following ?20 ??M ?TPEN ?treatment) ?in ?MDA-MB-231 cells13. Co-treatment of MDA-MB-231 ?cells ?with ?TPEN ?(20 ??M) ?plus ?zinc  25 (10, ?20 ?& ?40 ??M) ?completely ?inhibited ?activation ?of ?caspase-3, providing further evidence that intracellular zinc status regulates caspase activation13.  Changes in the concentration of intracellular free calcium (Ca2+) have also been shown to have an important role in regulating the intrinsic mitochondrial apoptotic pathway13,151. Recent research from ?our ?laboratory ?indicated ?that ?TPEN ?(20 ??M)-induced zinc depletion was associated with a small sustained rise in intracellular calcium ions in MDA-MB-231 cells (13% at 3 h)13. Blocking the mitochondrial calcium uniporter inhibited TPEN-induced caspase-3 activity, indicating that mitochondrial calcium uptake mediates apoptosis151. Chelation of intracellular calcium during TPEN treatment also increased the mitochondrial membrane potential, as well as inhibiting cytochrome c release, caspase-9 activity and caspase-3 activity13. Calcium chelation (20 & 40%) was able to partially inhibit DNA fragmentation (31 and 58%, respectively at 48 h)13. This research points to a role for calcium as a regulator of the mitochondrial mediated apoptosis, specifically cytochrome c translocation and the caspase-dependent pathway.  Additionally, a study on the role of zinc depletion in VSMCs also implicated intracellular calcium flux in modulating apoptosis122. Addition of plasma from zinc-deficient rats to VSMCs resulted in a sustained elevation of intracellular calcium and was associated with increased apoptosis. Calcium chelation prevented induction of apoptosis in this model. Treatment with of VSMCs with the zinc-deficient plasma was also associated with activation of BCL-2-associated death promoter protein (BAD), a pro-apoptotic protein, via dephosphorylation. Inhibition of calcineurin, a calcium-protein phosphatase, decreased BAD dephosphorylation and prevented apoptosis. Since calcineurin is activated by calcium, the  26 rise in calcium following zinc deficiency may regulate mitochondrial membrane permeability through its effects on BAD phosphorylation. Zinc depletion-induced apoptosis has also been linked to stability of the X-linked inhibitor of apoptosis protein (XIAP).  XIAP is an important regulator of both the initiation and execution phases of apoptosis, as it strongly inhibits both caspase-3 and caspase-9152. In MDA-MB-231 ?breast ?cancer ?cells, ?zinc ?depletion ?using ?TPEN ?(10 ??M) ?quickly ?resulted ?in ?loss of full length XIAP after 3 h153.  This was followed by activation of caspase-3 (7 fold increase in activity) and PARP cleavage after 24 h TPEN treatment. Similarly, in prostate cancer PC-3 cells, 16 h TPEN treatment (5 ?M) resulted in loss of XIAP and PARP cleavage. Zinc is known to be structurally important for XIAP, however the mechanism by which zinc depletion causes breakdown of XIAP is not well understood153.  Recent studies have indicated that zinc depletion-induced apoptosis may also involve release of AIF from the mitochondria, however, the importance of AIF in mediating zinc depletion-induced apoptosis is still unclear. Mendivil-Perez ?et ?al. ? found ? that ?TPEN ?(3 ??M, ?24h) treatment promoted nuclear translocation of AIF, in addition to caspase-3 activation in Jurkat T leukemia cells154. Guo et al. reported an elevated level of AIF in the cytosol of mouse osteoblastic MC3T3-E1 ?cells ? following ?TPEN ?treatment ? (5 ??M, ?24h) ? in ?addition to activation of caspase-3 and -9149. Pretreament of MC3T3-E1 cells with a broad range caspase inhibitor only partially protected cells from zinc depletion-induced apoptosis. Pretreatment of human colorectal carcinoma LIMI215 cells with a caspase-3 inhibitor or a broad range caspase inhibitor, followed ? by ? TPEN ? (25 ? ?M) ? treatment, ? resulted in partial reduction of DNA fragmentation150. These studies indicate that AIF-mediated / caspase-independent  27 apoptotic pathways may play a role in zinc depletion-induced apoptosis. However, caspase inhibition ? mostly ? suppressed ? DNA ? fragmentation ? in ? TPEN ? (15 ? ?M)-treated PBL cells, indicating that AIF did not play a major role in inducing apoptosis in this cell line137. Therefore, the importance of caspase-independent pathways in zinc depletion-induced apoptosis may vary in a cell-type specific manner. Zinc deficiency has also been found to increase oxidative stress in many studies in vivo and in vitro, which may play a role in zinc depletion-induced apoptosis109. Zinc stabilizes cell membranes and macromolecules, protecting them from oxidative stress109. Zinc itself is redox inert in biological systems, but it indirectly exerts antioxidant effects155. The antioxidant effects of zinc are not well understood, but may be include a metallothionein antioxidant function and zinc-dependent proteins / functions in the mitochondrial electron transport chain155. Oxidative damage is a well-known activator of apoptosis109, therefore zinc may protect against oxidative stress-induced apoptosis. In fact, a number of studies showed that antioxidant supplementation was able to suppress zinc depletion-induced apoptosis. For example, zinc-deficiency in rats was associated with increased expression of inducible nitric oxide synthase (NOS), and treatment with a NOS inhibitor suppressed zinc-deficiency induced intestinal damage, inflammatory skin lesions and apoptosis in the intestines and skin156,157. In cultured rat VSMCs, pre-incubation of cells with N-Acetyl-L-Cysteine (NAC), an antioxidant, reduced oxidative stress and prevented the induction of apoptosis following addition of plasma from zinc deficient rats122. Treatment of breast cancer cells (MCF-7, MDA-MB-468) ?with ?NAC ?also ?inhibited ?loss ?of ?viability ?induced ?by ?TPEN ?(10 ??M) ?and ?DTPA ?(100 ??M) ?14. In Jurkat T leukemia cells, addition of NAC not only protected against TPEN ?(3 ??M) ?induced ?apoptosis, ?but ?also ?rescued ?cells ?from ?apoptosis ?after ?6 ?h TPEN  28 treatment154. ?Treatment ?of ?rat ?hepatocytes ?with ?NAC, ?did ?not ?prevent ?TPEN ?(30 ??M) ?induced caspase-3 activation at 10 h, but it did block apoptosis158. Zinc depletion in MDA-MB-231 ?cells ?(20 ??M ?TPEN) ?increased ?oxidative ?DNA ?damage, and was associated with increased production of inducible NOS and reactive nitrogen species159. Furthermore, inhibition of NOS, reduced caspase-3 activity by one third.  Zinc may also regulate apoptosis through transcriptional gene regulation. In human cervical HeLa cancer cells, only 3 hours of TPEN ?treatment ?(30 ??M) ?inhibited ?the DNA-binding activity of the specificity protein (Sp) family of transcription factors and 6 hours of TPEN treatment resulted in cleavage of the Sp transcription factors132. TPEN treatment (30 ?M ?TPEN, ?24h) ?was ?also ?associated ?with ?loss ?of ?DNA ?binding ?activity ?of ?Zn3-Sp1 to its cognate DNA site (sodium glucose co-transporter 1 gene) in LLC-PK1 pig kidney cells134. In Jurkat ?T ?leukemia ?cells, ?TPEN ?(3 ??M) ?induced ?apoptosis ?was ?associated ?with ?activation ?of ?NF-kB and c-Jun transcription factors, which were found to regulate loss of mitochondrial membrane potential, plasma membrane integrity and development of apoptotic nuclei154. Therefore, zinc depletion causes alterations in transcription factor activity, which may play a role in regulating apoptosis; however this has not been well studied.   In summary, zinc depletion has been shown to induce apoptosis in a wide range of cells and tissues, in a concentration and cell type-dependent manner.  Zinc depletion-induced apoptosis involves increased intracellular calcium-induced release of cytochrome c from mitochondria and activation of caspases.  This body of evidence suggests that that zinc depletion induces apoptosis through the intrinsic mitochondrial pathway, such as in the case  29 of MDA-MB-231 breast cancer cells; however, the exact mechanisms whereby zinc depletion induces apoptosis remains to be elucidated. 1.4. MicroRNAs: Regulators of Apoptosis 1.4.1. Introduction to microRNA MicroRNAs (miRs) are a recently discovered class of short, non-coding RNAs, that regulate numerous metabolic processes by post-transcriptional gene regulation15. MiR research is rapidly evolving as the first miR was only identified in 1993160,161. The first functional role for a miR in mammalian development was discovered in 2004162. Since then the field has burgeoned, with over 2500 mature miRs reported in humans163.  MiRs are short, non-coding RNAs of 18-25 nucleotides in length, which regulate numerous metabolic processes by post-transcriptional gene regulation15. MiR regulate target messenger RNA (mRNA) through complementary base pairing, typically resulting in degradation of target mRNA164. MiR have wide-ranging physiological roles including embryogenesis, hematopoiesis, muscle development and immunity165,166.  The level of gene regulation by miRs is extensive and complex as each miR may regulate a few hundred target mRNAs, and multiple miRs may coordinate regulation of a single mRNA transcript167. In fact, it is estimated that more than 60% of human mRNAs contain conserved miR target sites, indicating widespread regulation by miRs168. MiR targets include enzymes and transcription factors, and miR-target interactions regulate key cellular activities including differentiation, proliferation and apoptosis169.   30 Despite considerable advances in miR research, there is still much that is unknown regarding the functionality of miR-mediated gene silencing, as well as regulation of miR expression and integration with cellular signaling pathways, etc.164,170.  1.4.2. MiR biogenesis A multi-step process, including nuclear and cytosolic processing, produces mature miR (Figure ). In the nucleus, long primary (pri-) miR sequences are encoded within introns, exons, and intergenic regions171. Pri-miR are transcribed mainly by RNA Polymerase II into long primary transcripts of variable length (often 3-4 kb)172,173. The pri-miR contains a stem loop or hairpin-like structure, formed by a double stranded RNA stem and an unpaired RNA loop, which eventually becomes the mature miR following cleavage at two site-specific events.  In the nucleus a multiprotein complex, known as the microprocessor, cleaves the pri-miRs174?177. The microprocessor complex contains a RNA-binding domain called Pasha/DiGeorge syndrome critical region 8 (DGCR8) for substrate recognition and recruitment of the RNAse enzyme Drosha175,176. Drosha cleaves the pri-miR into shorter precursor (pre-) miR of about 70 nucleotides in length178,179. Subsequently, pre-miRs are exported from the nucleus by a carrier protein called Exportin180?182. In the cytoplasm, pre-miR are processed by the miR generating complex containing a RNase enzyme, Dicer, producing double-stranded mature miR of approximately 22 nucleotides in length183. The miR generating complex may contain additional proteins which enhance cleavage of pre-miRs to mature miRs. These include trans-activating response RNA-binding protein (TRBP) and protein activator of PKR (PACT)184.   31 Although the pathway described was once thought to universally describe miR biogenesis, recent research reveals many variations in this pathway and the existence of individual miR-specific processing alterations185. For example, miR may undergo processing independent of Drosha or Dicer185,186. 1.4.3. MiR function For miRs to exert their function, they are incorporated into an effector RNA-induced silencing complex (RISC) containing Dicer, TRBP, and Argonaute (Ago)185. In humans there are four Ago proteins (Ago 1-4), which mediate miR-targeted silencing of protein expression. One of the mature miR strands is transferred to an Ago protein by two chaperone proteins (Hsc70/Hsp90)187, while the other strand is degraded188. The thermodynamic stability of the miRNA duplex is an important determinant of strand selection, with the miR that is less ?stably ?base ?paired ?at ?the ?5? ?end ?being ?more ?likely ?to ?be ?loaded ?in ?the ?RISC ?complex188,189. Sometimes multiple mature miRs arise from a single precursor, and in this case ?the ?5? ?arm ?and ?3? ?arms ?are ?distinguished ?by ?annotation ?with ?-5p and -3p.  The RISC mediates degradation of mRNA based on complementarity between a miR and its targeted mRNA. The mature miR contains a seed region of 6-8 ?nucleotides ?at ?the ?5? ?end, which binds to complementary base pairs in the target mRNA164. The seed region may bind to any part ?of ?the ?mRNA, ?but ?binding ?to ?the ?3? ?untranslated ?region ?(UTR) ?is ?the ?most ?frequently documented and this tends to decrease target mRNA expression164. Target mRNAs are silenced by deadenylation and subsequent degradation, or translational inhibition, but the mechanisms involved are not well understood170,190. Recent studies  32 suggest that degradation of miR-targeted mRNAs is the primary type of gene silencing by miRs in mammalian cell cultures170.   The canonical model of miR-mediated gene regulation is that of negative regulation via ?miR ?binding ?to ?the ?3? ?UTR ?of ?the ?target ?mRNA18. However, miRs have also been found to positively regulate gene expression. For example, during cell cycle arrest induced by serum starvation, two miRs (miR-369-3 & let-7) upregulated translation of their targeted mRNAs ?by ?binding ?to ?their ?3? ?UTRs191. MiR-10a was also found to activate expression of ribosomal ?protein ?mRNAs ?by ?binding ?to ?their ?5? ?UTR192. The mechanisms involved in the regulation of gene expression by miRs are currently not well understood and are still controversial164. 1.4.4. MiR stability MiRs are typically very stable and the majority of mature miRs persist for hours up to days in the cell193,194. In human embryonic kidney (HEK)-293T cells, the vast majority (95%) of miRs remained stable for at least 8 h after inhibiting miRs transcription with a chemical inhibitor195. In another experiment, miR turnover was measured after loss of Dicer enzymatic activity in murine primary bone marrow derived macrophages196. The miRs investigated were found to have half-lives ranging from 28 to 211 h (~9 days). Therefore, miRs are generally quite stable; but the stability of miR varies on an individual miR basis.  MiR stability may be affected by a number of factors including the cell cycle stage, growth factors and the presence of miR degrading enzymes194.  33 1.4.5. MiR expression in breast cancer Our understanding of the role of miR in the development and progression of breast cancer is in its infancy; however, it has been established that miRs are involved in the regulation of every known cellular process related to cancer, such as differentiation, proliferation and apoptosis197. Abnormal miR expression profiles have been reported for various types of cancer, including breast cancer198?201. Unique miR expression profiles or ?fingerprints? ?are ?associated ?with ?estrogen ?receptor ?(ER) status, tumor differentiation, invasiveness and response to therapy198,200,201. MiR dysregulation in cancer may occur due to a number of factors including alterations in miR genomic copy number and localization, epigenetic regulation, transcription factor activity and miR processing202. On a global scale, miR expression was found to be generally downregulated in a broad range of cancer tissues and cancer cell lines when compared to normal tissues203. A large study by Devinge et al., involving 1,302 breast cancer samples as well as 28 breast cancer cell lines, compared to 116 normal breast tissue samples showed that mature miR expression was globally decreased in breast cancer tissue and cell lines compared to normal tissue204. Furthermore, expression of the global miR population gradually declined as the tumor grade increased and was the lowest in breast cancer cell lines. Decreased expression of miRs in cancer cells and tissues may occur through alterations in miR processing. Assessment of pri-miR and mature miR expression in a wide range of primary tumors and normal tissue samples, indicated that the level of pri-miR did not correspond to the reduced expression of mature miR, indicating that global downregulation of miR expression in cancer tissues may occur at the post-transcriptional level205. In contrast, expression of pri- and mature miR were positively correlated in normal  34 tissue samples. Another experiment examining the correlation between miR precursors (both pri- and pre-miRNA) compared to mature miRs in 37 human cancer cell lines also found that there was little correlation between mature and precursor miR expression206. The study showed that some miR precursors were retained in the nucleus, which would prevent cytosolic processing to mature miR. A panel of 22 normal tissues tested showed a higher correlation between precursor and mature miRNA expression. Breast cancer has been associated with alterations in the miR processing machinery, which could play a role in the abnormal miRNA expression observed in breast cancer. One study found that in 18% of breast cancer cases, Drosha was downregulated, which was associated with breast cancer characteristics including high grade, high proliferation index, lack of BCL-2 expression and overexpression of human epidermal growth factor receptor type II (HER2)207. Dicer was also downregulated in 46% of breast cancer cases207. Decreased expression of Dicer is more likely to occur in ER negative breast cancer cells201,207?209. Dicer downregulation was also associated with some other cancer characteristics including lack of expression of progesterone receptor (PR) and BCL-2, as well as high grade and proliferation index207. In the previously mentioned study by Dvinge, et al., Dicer expression was only mildly reduced in the higher-grade tumors, which could not account for the observed global downregulation in miR expression204. This study also found that expression of other proteins involved in miR stability varied by tumor grade. The terminal uridyltransferase 4 / zinc-finger, CCHC domain-containing protein 11, involved in miR inactivation, was slightly upregulated and the polyadenylate polymerase associated domain containing 4 / Gld-2, involved in miR stabilization, was mildly downregulated.  35 Therefore, these enzymes may also play a role in regulating miR expression in the context of breast cancer. Inhibition of global miR expression may aid cancer development. Blocking miR maturation with inhibitors of Drosha, DGCR8, and Dicer1, increased transformation in mouse (LKR13) lung cancer and human cancer cell lines (i.e. MCF-7 breast cancer, U2OS osteosarcoma, and HCA7 colorectal cancer), as well as murine lung tumorigenesis in vivo210.  While the functional roles of the majority of miRs have not yet been determined, some miRs have been found to regulate breast cancer development211. Individual miRs may play either oncogenic or tumor suppressive roles in breast cancer. A few select examples will be described in more detail below, providing support for the key roles of miRs in modulating the course of breast cancer. For instance, oncogenic miR-21 is the most commonly upregulated miR across many types of cancer, including breast cancer212. Inhibition of miR-21 expression in MCF-7 breast cancer cells reduced cell growth in vitro as well as in vivo in a xenograft mouse model213. MiR-21 has been found to target the tumor suppressors programmed cell death 4214,215 and tropomyosin 1 proteins216 to promote cell transformation in MCF-7 breast cancer cells.  In another study, a genetic screen was used to identify miRs involved in metastasis217. Upregulation of miR-373 and miR-520c were identified as promoting migration in vitro in MCF-7 breast cancer cells, as well as tumor invasiveness in vivo in immunodeficient mice injected with MCF-7 cells. Investigation of miR-373 expression in clinical samples also indicated that miR-373 was upregulated in breast cancer metastasis. Downregulation of tumor suppressor miRs may also aid breast cancer pathogenesis. For example, the let-7 family was reported as poorly expressed in breast cancer stem cells,  36 known as breast tumor-initiating cells (BT-IC) and was found to regulate self-renewal and differentiation of breast cancer cells218. Upregulation of let-7 reduced tumor formation in vivo in immunodeficient mice injected with a highly malignant breast cancer cell line enriched with BT-IC (Sk-3rd).  Downregulation of miR-132 may also play a role in breast cancer development. Reduced abundance of miR-132 was reported in human clinical samples of breast ductal carcinoma in situ (DCIS), an early stage of breast cancer, compared to paired samples of adjacent normal tissue219. Upregulation of miR-132 in MCF-7, MDA-MB-231 and BT549 breast cancer cell lines decreased cell viability and inhibited colony formation.  In summary, abnormal expression of miRs is implicated in breast cancer and targeting miR expression alters breast cancer development. The next section will discuss the role of miRs in regulating apoptosis in breast cancer cells. 1.4.6. MiRs & apoptosis in breast cancer Impaired apoptosis is one of the hallmarks of cancer cells110,220. In breast cancer cells, abnormal miR expression can reduce susceptibility to apoptosis, thus contributing to increased survival of breast cancer cells17. MiRs exert their effects by regulating many proteins involved in cell death and survival pathways in cancer cells16. A number of miRs have been found to target the intrinsic mitochondrial apoptotic pathway, particularly at the level of the BCL-2 family, which in turn regulates mitochondrial membrane permeability17.    For example, miR-21 is a potent oncogene with antiapoptotic effects212. Inhibition of miR-21 in MCF-7 breast cancer cells as well as a MCF-7 derived xenograft carcinoma in  37 mice, suppressed growth of breast cancer cells due to decreased cell proliferation and increased apoptosis213. Interestingly, treatment of MCF-7 cells with a broad range caspase inhibitor, prevented the growth inhibitory effects of miR-21 knockdown, implicating caspases as effectors in miR-21-induced inhibition. Furthermore, inhibition of miR-21 in MCF-7 cells and MCF-7 derived breast tumors resulted in impaired expression of BCL-2, an anti-apoptotic protein, suggesting that miR-21 may exert its oncogenic effects through the death inhibitory functions of BCL-2.  Another miR generally upregulated in breast cancer tumors is miR-155, but in a cancer type specific manner198,199,221. MiR-155 expression has been found to regulate breast cancer cell survival and growth. In human invasive ductal breast carcinoma BT-474 cells, miR-155 was expressed at lower levels (compared to a number of other breast cancer cell lines) and upregulation of miR-155 expression in BT-474 cells promoted cell proliferation and suppressed apoptosis221. Conversely, fibroblast breast cancer HS578T cells had higher miR-155 expression, and knock down of miR-155 inhibited cell proliferation and induced apoptosis. MiR-155 directly targeted the pro-apoptotic transcription factor forkhead box O3a (FOXO3a) through translational inhibition. Reduced FOXO3 expression in turn inhibited the expression of its downstream targets Bim (a pro-apoptotic member of the BCL-2 family) and p27(Kip1, a cell cycle inhibitor) to prevent apoptosis. Expression of miR-155 also conferred resistance to apoptosis induced by multiple chemotherapeutic agents (i.e. doxorubicin, VP16, and paclitaxel). Other studies also suggest that altered miR expression in breast cancer can provide resistance to chemotherapeutic drug-induced apoptosis. For example, the miR-221/222  38 cluster was found to be upregulated in ER- cell lines and breast tumors222. Furthermore, miR-221/222 were shown to directly negatively regulate ER and to confer resistance to tamoxifen treatment in vitro in MCF-7 and T47D cells222. Upregulation of miR-221/222 blocked tamoxifen-induced apoptosis in MCF-7 by targeting the cell cycle inhibitor p27(Kip1)223.  Increased expression of miR-125b provided resistance to paclitaxel (Taxol)-induced apoptosis in breast cancer MDA-MB-231 and MDA-MB-435 breast cancer cells224. MiR-125b upregulation delivered Taxol resistance by suppression of caspase-3 activity in MDA-MB-435 cells. Further investigation revealed that miR-125b exerted pro-survival effects by negatively regulating the pro-apoptotic protein BCL-2 antagonist killer 1 (BAK1) in multiple breast cancer cell lines, specifically MDA-MB-435, MDA-MB-231, MCF-7 and SkBr3 cells. BAK1 is a critical regulator of apoptosis as it induces pore formation in the mitochondria. Inhibition of BAK1 substantially restricted the ability of Taxol to induce apoptosis in breast cancer MDA-MB-435 and MDA-MB-231 cells. Also, increasing BAK1 expression in cells with upregulated miR-125b, resulted in restored sensitivity to Taxol treatment. Taken together, these findings indicate that miR-125b-mediated negative regulation of BAK1 provides an important mechanism for its resistance to Taxol-induced apoptosis. Although the previous examples provide evidence of the anti-apoptotic effects of some highly expressed oncogenic miRs, downregulation of pro-apoptotic miRs may also contribute to breast cancer pathogenesis. In fact, globally the majority of miRs are  39 downregulated in cancers203, and impaired miR expression has been found to promote tumorigenesis210.  For example, invasive ductal breast carcinomas displayed low levels of miR-497 compared to paired normal breast tissues, and its expression was inversely correlated to lymphatic metastasis, tumor size and presence of HER-2225. Further investigation revealed that miR-497 inhibited cellular growth in MCF-7 breast cancer cells, by causing G0/G1 cell cycle arrest and inducing apoptosis. Upregulation of miR-497 suppressed the expression of Bcl-w, an anti-apoptotic member of the BCL-2 family.  In MCF-7 cells, miR-195, miR-24-2 and miR-365-2 were identified as negative regulators of the BCL-2 oncogene through a combination of computational predictions and experimental analysis226. Upregulation of these three miRs caused loss of mitochondrial membrane potential, release of cytochrome c, activation of caspase-9 and induction of apoptosis. Furthermore, overexpression of these miRs acted synergistically with the chemotherapeutic drug etoposide, to increase apoptosis in MCF-7 cells.   In summary, miRs targets include both pro- and anti-apoptotic proteins. Altered miR expression has been shown to regulate apoptosis and sensitivity to anticancer therapies in breast cancer cells. Therefore, targeting miRs could be a novel therapeutic strategy for treating breast cancer, as well as other cancers18.    40 1.5. Zinc and MiR Expression Regulation of miR expression by nutritional means, in particular by zinc status, is a newly emerging area of research with relatively few studies to date. Recently, the effects of low dietary zinc intake by young males on the serum miR profile was investigated, to determine ?if ?a ?molecular ?miR ??signature? ?could ?be ?detected19.  Following low zinc intake (10 d), serum zinc decreased and the miR expression profile was altered; of the 88 miRs tested, 20 had a greater than 1.5 magnitude fold change. After dietary zinc repletion, nine miRs responded in an opposite manner to zinc depletion, indicating that they may serve as useful biomarkers of zinc status.  Chronic zinc deficiency was investigated in a rat esophagus cancer model and shown to alter the miR profile in all tissues investigated, which were the esophagus, skin, lung, pancreas, liver, prostate and peripheral blood mononuclear cells20. In particular, two oncogenic miRs: miR-21 and miR-31 were the most commonly upregulated across the tissues profiled. Further research, using an esophageal cancer model in rats, revealed that miR-21 and miR-31 responded in a reversible manner to dietary zinc intake. Expression of these two miRs was upregulated in tumor bearing zinc deficient rats, but dietary zinc replenishment prevented cancer formation and reduced expression of miR-21 and miR-31, to levels similar to the control group. In vitro studies also point to zinc as a regulator of miR expression. In human breast cancer MCF-7 cells, zinc supplementation plus Clioquinol, a zinc ionophore, increased intracellular zinc content to cytotoxic levels, which was associated with global downregulation of miR21.  Zinc cytotoxicity suppressed Dicer and Ago2 protein expression,  41 which are used to produce and stabilize mature miR, respectively. Clioquinol plus zinc also increased assembly of processing bodies (P-bodies), which are small cytoplasmic granules where it is thought that RISC mediated regulation of mRNA may take place.   A recent study investigated the effects of zinc treatment on expression of some miRs in human prostate cancer 22Rv1, PC-3, and LNCaP cells and a non-tumorigenic human prostate PNT1A22.  MiR-23a was elevated in all four cell lines following zinc treatment, while other miRs displayed cell-type specific trends. Altered miR expression may play a role in zinc homeostasis in prostate cancer, which is associated with reduced zinc. The miR-183-96-182 cluster was overexpressed in prostate tumors and shown to regulate zinc homeostasis in primary prostatic epithelial (PrE) cells, as well as human embryonic kidney HEK-293 cells227. Overexpression of the miR-183-96-182 cluster lowered intracellular zinc content and decreased expression of six zinc transporters (Zip1, Zip3, Zip7, Zip9, ZnT1, and ZnT7) in PrE cells. Increased expression of the miR-183-96-182 cluster also decreased zinc uptake in HEK-293 cells. Therefore, dysregulation of miRs may contribute to altered zinc homeostasis. Currently, there are no published studies on the role of miRs in zinc depletion-induced apoptosis. More research is still needed to determine the functional significance between zinc status and miR expression, especially in the context of cancer development and progression.   42  Figure 1.1: Key elements of the intrinsic mitochondrial apoptotic pathway.  An apoptotic signal induces permeabilization of the outer mitochondrial membrane (OMM), causing release of cytchrome c. In the cytosol, cytochrome c binds apoptotic protease activating factor (Apaf1), forming the apoptosome. The apoptosome activates caspase-9, which in turn activates caspase-3. Caspase-3 cleaves the inhibitor of caspase-activated deoxyribonuclease (ICAD), resulting in DNA fragmentation and apoptosis.13 .   43  Figure 1.2: General pathway for microRNA (miR /miRNA) biogenesis and function.  Primary (pri-) miRs are transcribed in the nucleus and undergo processing by Drosha into precursor (pre-) miRs, followed by Exportin-5 mediated nuclear export. In the cytoplasm, pre-miRs undergo an additional processing step by Dicer, producing double stranded mature miR. One of the strands of mature miR is loaded into the RNA-induced silencing complex (RISC) to mediate translational repression or degradation of target mRNA.202   44 1.6. Hypothesis Zinc depletion alters miR expression in human breast cancer MDA-MB-231 cells, which plays a role in mediating zinc depletion-induced apoptosis. 1.7. Overall Objective and Specific Aims The overall objective of this project was to determine the involvement of miRs in zinc depletion-induced apoptosis in MDA-MB-231 cells.  The specific aims of this research project were:  1) To determine if zinc depletion induced apoptosis in human breast cancer MDA-MB-231 cells, and 2) To profile the effects of zinc depletion on miR expression in human breast cancer MDA-MB-231 cells. 45 Chapter 2: Zinc depletion-induced apoptosis is associated with altered microRNA expression in human breast cancer MDA-MB-231 cells 2.1. Materials and Methods 2.1.1. Cell culture system Human breast cancer MDA-MB-213 cells (American Type Culture Collection, Manassas, Virginia) were routinely cultured ?in ?Dulbecco?s ?Modified ?Eagle?s ?Medium ?(DMEM; Sigma, St Louis, Missouri) supplemented with fetal bovine serum (FBS, 10%; Gibco, Grand Island, New York), sodium pyruvate (1 mM; Gibco), penicillin-streptomycin (50 ?units/ml ?and ?50 ??g/ml, ?respectively;? Gibco) at 37?C, 10% CO2. Through out the rest of this ?thesis, ?this ?medium ?is ?referred ?to ?as ?the ??regular ?medium?. ?The ?cells ?were ?cultured ?in ?10 ?cm Petri dishes at an initial density of 5 x 105 cells / dish. Cells were harvested for passage when the plates reached 80 - 95 % of confluence. Cells with a passage number of 35-45 were used in this research project. 2.1.2. Depletion of intracellular zinc N,N,N?,N?-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN; Sigma) stock solution was prepared by dissolving in dimethyl sulfoxide (DMSO; Sigma) with a concentration of 10 mM. The stock solution was stored at -20?C and thawed at room temperature prior to use.  Prior to zinc depletion, MDA-MB-231 cells were cultured in the regular medium for 3 days at 37?C, 10% CO2 with an initial density of 2.5 x 105 cells per 6 cm dish. The regular  46 medium was replaced 2 days after initial seeding. At the end of the 3 day growth period, the cells were treated with TPEN, by adding the 10 mM stock TPEN solution to the regular with an equal volume of DMSO. The cells were then cultured at 37?C, 10% CO2 for 0 - 72 h, depending on the experiment. 2.1.3. Apoptosis assay Apoptotic cells were measured by propidium iodide (PI; Sigma) flow cytometric assay228. PI binds to the DNA stoichiometrically, providing a measurement of the total cellular DNA content. In late-stage apoptotic cells, fragmented DNA leaves the cells resulting in lower DNA content. Apoptotic cells therefore are characterized by the presence of a sub-G1 hypodiploid peak. MDA-MB-231 cells were cultured in the growth medium with an initial density of 2.5 x 105 cells/well in a 6 cm plate. The growth medium was replaced after 2 days. At the end ?of ?the ?3 ?day ?growth ?period, ?cells ?were ?treated ?with ?TPEN ?(10 ??M) ?or ?DMSO ?(control) ?for 0, 3, 6, 12, 24, 48, or 72 h. After the treatment period, the medium containing unattached cells was removed from both the control and TPEN plates and was transferred to a 15 ml conical tube. The cell culture plates containing adherent cells were rinsed once with warm phosphate buffered saline (PBS; 37?C). Adherent cells were harvested by addition of warm 0.25% trypsin-EDTA (ethylenediaminetetraacetic acid, Gibco; 37?C) and incubated (37?C, 10% CO2) for approximately 3 min or until cells had unattached, followed by addition of an equal volume of regular cold medium. The harvested cells were then placed into the same  47 tube used to collect the medium containing unattached cells. Cells were pelleted by centrifugation (300 g, 4?C, 5 min) and the supernatant was removed. The cells were washed once by resuspension in PBS (1 ml) by pipetting briefly, followed by centrifugation (300 g, 4?C, ?5 ?min) ?and ?removal ?of ?the ?supernatant. ?The ?cells ?were ?fixed ?by ?slowly ?adding ?100 ??l ?cold ?PBS ?(4?C), ?followed ?by ?900 ??l ?of ?cold ?ethanol ?(-20?C; 70% EtOH in H2O, v/v) while gently vortexing, and then left at 4?C overnight. After fixation, the cells were pelleted by centrifugation ?(400 ?g, ?5 ?min, ?4?C), ?washed ?once ?with ?PBS ?(500 ??l), ?re-pelleted by centrifugation (400 g, 5 min, 4?C) and the supernatant was removed. The cell pellet was then re-suspended ?in ?DNA ?staining ?solution ?[200 ??g/ml ?ribonuclease ?A ?(Sigma) ?and ?20 ??g/ml ?PI ?(Sigma) in PBS; pH 7.4] at approximately 1 ml / 106 cells. Finally, the cell suspension was transferred to a 5 ml Falcon tube and incubated at room temperature and in the dark for 30 min. The presence of PI-stained cells was determined by flow cytometry (BD FACSCalibur Flow Cytometer and CellQuest Pro Software; BD Biosciences, San Jose, California). The excitation and emission wavelengths were 488 nm and 610 nm, respectively. A total of 25,000 events were counted for each sample. The percentage of cells with fragmented DNA content or apoptosis was determined using FlowJo Software (version 10.0.4; Tree Star, Ashland, OR). 2.1.4. Total RNA isolation  MDA-MB-231 cells were cultured under the same conditions described previously, and ?the ?cells ?were ?treated ?with ?TPEN ?(10 ??M) ?or ?DMSO ?only (control) for 3, 12 or 24 h. At the end of the treatment period, total RNA was extracted using the mirVANA miR isolation kit (Ambion, Life Technologies Corporation, Burlington, Ontario) according to the  48 manufacturer?s ?instructions. ?Briefly, ?the ?medium ?containing ?floating ?cells ?was ?collected ?by ?transferring to a 15 mL conical tube on ice and then pelleted by centrifugation (600 rpm, 4?C, 5 min). Both floating and adherent cells were rinsed with cold PBS and the denaturing lysis buffer was added in proportion to the amount of cells (100 ? 600 ??l). ?A ?spatula ?was ?used to dislodge adherent cells, which were then pipetted into a 2.0 mL microcentrifuge tube. The homogenate was added to both floating and adherent cells at 1/10th of the lysate volume and the mixture was vortexed briefly, followed by incubation for at least 10 min on ice. The lysate mixtures from the floating cells and adherent cells were combined into a single tube and then mixed with an equal volume of acid phenol-chloroform, followed by vigorous vortexing for two minutes. Samples were centrifuged (16,100 g, room temperature, 5 min) and the upper aqueous phase was carefully removed with minimal disturbance to the interface. Then 1.25 volumes of room temperature ethanol was added to the aqueous phase and mixed thoroughly by inversion. Subsequently, the samples were precipitated onto the filter cartridge provided in the RNA isolation kit. The RNA was purified using the provided wash ?solutions ?according ?to ?the ?manufacturer?s ?instructions. ?Total ?RNA ?was ?collected ?by ?elution with 0.1 mM EDTA in nuclease-free ?water ?(100 ??l) ?heated ?to ?95?C. ? RNA quality was assessed by both purity and integrity. RNA purity was measured using the Nanodrop spectrophotometer 260/280 and 260/230 ratios (Thermo Fisher Scientific, Wilmington, Delaware) and RNA integrity was visualized using gel electrophoresis (1% agarose gel). RNA samples having a 260/280 ratio of > 1.8 (1.99 ? 2.49), a 260/230 ratio of > 1.0 (1.01 ? 3.07) and being free of degradation were used for miR expression profiling. Intact RNA samples with 260/280 ratio > 1.8 (2.02 ? 2.08) were used for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay.  49 The majority of RNA samples used for qRT-PCR had a 260/230 ratio of > 1.0 (1.01 ? 3.23), except for 2 samples with lower ratios (3hT: 0.88, 12hT: 0.57).  2.1.5. MiR microarray assay The genome-wide human (noted by the hsa prefix) miR profile was assessed through microarray profiling provided by LC Sciences (Houston, Texas; n = 3). Profiling was completed for all mature miRs included in the miRBase (a miR repository) release 19 (August 2012), containing 2019 mature human miRs. Starting with a total RNA sample (4?8 ?g), RNAs were extended with a poly(A) tail and ligated to an oligonucleotide tag for later fluorescent dye staining. RNA samples were hybridized to detection probes overnight on a ?Paraflo microfluidic chip (Atactic Technologies, Houston, Texas)229,230. Each detection probe was composed of a chemically modified oligonucleotide, which was complementary to target miR (from miRBase) or other RNA (control), in addition to a polyethylene glycol spacer segment. The probes were prepared by in situ synthesis using photogenerated reagent chemistry and contained chemical modifications to balance the hybridization melting temperatures. Hybridization was performed at 34?C in 100 ?L of 6x saline-sodium phosphate-EDTA (SSPE) buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide. Each sample was analyzed on an individual chip containing four repeat probes per miR. Following hybridization, cyanine 3 dye was circulated through the chip for fluorescence staining. The microarrays were scanned with a GenePix 400B laser scanner (Molecular Devices, Sunnyvale, Califormia) and image digitization was performed using Array-Pro image analysis software (Media Cybernetics, Rockville, Maryland). Following background subtraction, data was normalized using the cyclic LOWESS (locally  50 weighted scatterplot smoothing) method231. The normalized signal intensity was reported on a relative scale from 0 ? 65,535. A minimum signal intensity of 32 for at least one individual sample was used as the cutoff for detectable miR expression.  2.1.6. MiR heat maps MiR expression was clustered according to similarities in gene expression across sample groups. Clustered heat maps were generated using z-scores of log-transformed signal intensities for each individual miR at the time point(s) evaluated. The distance metric used to determine similarity in gene expression was Euclidean distance, which is the distance between two points. Hierarchical clustering was performed based on average linkage, where the similarity of two clusters was determined from the average distance of all pairwise comparisons. Multiexperiment Viewer software (TM4 MicroArray Software Suite) was used to perform cluster analysis. 2.1.7. qRT-PCR miR assay Expression of some of the miRs most significantly affected by TPEN treatment (miR-132-3p, miR-1246, miR-1273g-3pg-3p, miR-4484, miR-4521 and miR-4787-5p) was subjected to validation using TaqMan qRT-PCR (n=6 with 2 technical replicates). The TaqMan miR assay is the gold standard for miR quantitation due to its high sensitivity and specificity over a wide range of miR expression levels232?234. Total RNA was quantified using a Qubit 2.0 Fluorometer (Life Technologies Inc., Burlington, ON). The starting amount of total RNA used in the RT reaction was 5 ng for miR-1246 and miR-1273g-3p, and 25 ng  51 for miR-132-3p, miR-4484, miR-4521 and miR-4787-5p. The RT and PCR reaction mixes were ?prepared ?according ?to ?the ?manufacturer?s ?instructions ?(Taqman ?MicroRNA ?Reverse ?Transcription Kit and TaqMan MicroRNA Assays; Applied Biosystems, Life Technologies Corporation). Briefly, a RT master mix was prepared containing 0.15 ??l ?100 ?mM ?dNTP ?(deoxyribonucleotide ?triphosphates) ?with ?dTTP ?(deoxythymidine ?triphosphate), ?1 ??l ?Multiscribe ?Reverse ?Transcriptase ?(50 ?U/?l), ?1.5 ??l ?Reverse ?Transcription ?Buffer ?(10x), ?0.19 ??l ?RNAse ?(ribonuclease) inhibitor ?(20 ?U/?l) ?and ?4.16 ??l ?nuclease-free water. The RT reaction (15 ul) was prepared with 7 ul master mix, 5 ul total RNA in nuclease-free water and 3 ?? ?RT ?Primer ?(5x). ?The ?RT ?reactions ?were ?run ?for ?30 ?min ?at ?16?C, ?30 ?min ?at ?42?C, ?and ?5 ?min at 85?C, and then cooled down to 4?C in an Eppendorf Mastercycler Gradient (Eppendorf AG; Hamburg, Germany). If the PCR experiment was not performed immediately after, the RT reactions were stored at -20?C. ?Briefly, ?the ?PCR ?reactions ?(20 ??l) ?consisted of 1 ?L TaqMan Small RNA Assay, 1.33 ?L cDNA, 10 ?L TaqMan Universal PCR Master Mix II (2x no UNG), and 7.67 ?L nuclease-free water. The PCR reactions were run with a 10 min incubation at 95?C followed by 40 PCR cycles of 95?C for 15 seconds and 60?C for 60 seconds in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). Relative quantification was determined according to the Delta Delta CT method235. MiR-16-5p was chosen as an endogenous control from 3 candidate endogenous controls screened. The microarray results indicated that miR-16-5p was highly expressed and that there were no significant differences with treatment, treatment duration and interaction (p < 0.05, Figure A.1, Table A.1). Expression of miR-16-5p was also subjected to validation by qRT-PCR. Relative quantification of miR-16-5p was determined according to the Delta  52 CT method235. Although the individual qRT-PCR runs showed some differences in miR-16-5p abundance, the differences were relatively small and did not indicate any consistent alterations in miR-16-5p expression at the different treatments or time points examined (Figure A.2-5). Overall, miR-16-5p was highly and consistently expressed, making it a suitable endogenous control. Furthermore, miR-16-5p has also been reported as a suitable endogenous control for malignant, benign and normal breast tissues in the literature236. 2.1.8. Statistics MicroArray statistics. The z-scores of log-transformed signal intensities were used for all statistical analyses. Data was analyzed for treatment effect at each time point using an error-weighted T-test. The effects of treatment, treatment duration, and their interactions among multiple time-points were analyzed using 2-way analysis of variance (ANOVA) (p < 0.05). The fold change standard deviation (SD) was calculated by propagation of error using the following formula237: ?? =1?1???2?1?????? + ????  ? = f(G1,G2) = G2/G1, where G1 and G2 are average values of group 1 and group 2 signal intensities ??, ???, and ??? are the standard deviations of ?, group 1 and group 2  53 qRT-PCR statistics. The effects of treatment, treatment duration, and their interactions on abundance of miR-16 (endogenous control) were analyzed by 2-way ANOVA, followed by post-hoc ?Tukey?s ?Honestly ?Significant ?Difference ?(HSD) ?test. ? Student?s ?t-test (p < 0.05) was used to assess significant differences between miR abundance ?in ?the ?control ?and ?TPEN ?groups ?at ?each ?time ?point. ?Welch?s ?t-test was applied when the variances between the control and TPEN groups were unequal (p < 0.05). One-way ANOVA was used to test the treatment-duration ?effects ?followed ?by ?Tukey?s ?HSD ?test ?for ?the significant difference among the 3 time points (p < 0.05). Outliers, which exceeded 3 standard deviations from the mean, were omitted from data analyses.  54 2.2. Results 2.2.1. Zinc depletion-induced apoptosis The time-course of zinc depletion-induced apoptosis in MDA-MB-231 cells is shown in Figure  as well as Figure A.6-12. The control group exhibited < 1% of apoptotic cells at all time points examined. At 0 h, 0.79 % of cells were apoptotic, indicating a low basal level of apoptosis ?in ?cultured ?cells. ?TPEN ?treatment ?(10 ??M) ?resulted ?in ?negligible ?levels ?of ?apoptosis ?after 3 and 6 h, which were comparable to the control group (0.5% vs. 0.4% at 3 h and 0.5% vs. 0.6% at 6 h for the control and TPEN groups, respectively). After 12 h TPEN treatment, only 1.2 % of cells had fragmented DNA, compared to 0.6% in the control group. By 24 h, the proportion of apoptotic cells increased slightly to 4.5%, versus 0.6% apoptosis in the control group. The longer TPEN treatment durations of 48 and 72 h substantially increased the proportion of apoptotic cells to 24.4 and 28.0% respectively, compared to 1.0 and 0.7 % in their corresponding controls. In summary, TPEN-induced depletion of intracellular zinc in MDA-MB-231 cells resulted in a time-dependent induction of apoptosis. After 24-h TPEN treatment, just under 5% of MDA-MB-231 cells were apoptotic, while TPEN treatment for 48 and 72 h substantially increased the proportion of apoptotic cells.   55 2.2.2. Zinc depletion altered miR expression The time-points chosen to investigate the effects of the early stages of zinc depletion-induced apoptosis on global miR expression were 3, 12 and 24 h, due to the low level of apoptosis (<5% cells with fragmented DNA) observed following up to 24 h TPEN treatment. The miR microarray detected 397 of 2019 unique mature human miRs in at least one of the samples (Table A.1), indicating that a subset of the human micoRNA genome (miRNome) was expressed in control and TPEN-treated MDA-MB-231 cells.  The global miR expression profile remained largely unchanged following three hours of TPEN treatment. The expression of just 8 miRs was significantly affected compared to the control group, with 6 miRs increased and 2 miRs decreased (Figure ). The signal intensity of miRs significantly affected after 3 hours TPEN treatment were generally low (< 500), with only one miR (182-5p) just exceeding a signal intensity of greater than 500 for at least one individual sample, which is the minimum signal required for validation by RT-qPCR. The expression of miR-182-5p was decreased by 1.3 times in the TPEN treated group compared to the control group (Table 2.1, Table A.1). Following 12 h TPEN treatment, the miR expression profile was largely affected with almost one-quarter of detected miRs significantly altered (90 / 397; Figure ). The expression of majority of differentially expressed miRs was increased (51 / 90). Of those miRs significantly affected, 10 miRs had a greater than two-fold change in expression and also met the minimum signal intensity cutoff of 500 for at least one individual sample (Table 2.2). Interestingly, the expression of all 10 of these miRs (miR-3127-5p, -5194, -4485, -132-3p, - 56 4734, -1273g-3pg-3p, -5096, -4484, -1973 and -4690-5p) was increased. Among these 10 miRs, miR-3127-5p and miR-5194 showed the greatest fold increase in the TPEN treated groups compared to their corresponding controls. However, the expression of miR-3127-5p and miR-5194 in the control groups was negligible, as the signal intensities were below the cutoff of 32 for detectable expression (average signal intensities: 1, 4, respectively, Table A.1). In the TPEN treated groups, there was a small increase in the signal intensities of miR-3127-5p and miR-5194 (average signal intensities: 490, 403 respectively). Therefore, the abundance of miR-3127-5p and miR-5194 was low in both the control and TPEN treated groups. The expression of miR-4485 and miR-132-3p was also very low in the control group (average signal intensities: 35, 38, respectively) and was significantly increased by TPEN treatment (average signal intensities: 1,217, 630, respectively), with a 34.5 and 16.4 fold increase, respectively. The remainder of the miRs displayed approximately 2-3 fold increases in their expression following 12 h TPEN treatment. After 24-h TPEN treatment, approximately one-quarter of miRs (94 / 397) were significantly affected, and the majority of differentially expressed miRs had increased expression (74 / 94; Figure ). Among those miRs having signal intensities greater than 500 for at least one individual sample and a fold change greater than two, the expression of 22 miRs was increased and 2 miRs was decreased (Table 2.3). The expression of 5 miRs (miR-132-3p, -1273g-3pg-3p, -1973, -4484 and -4485) was increased by more than two-fold after both 12 and 24 h TPEN treatment. There were numerous miRs significantly altered by zinc depletion, duration of zinc depletion, and their interactions. In total, 285 unique miRs were significantly affected by zinc  57 depletion, duration of zinc depletion and their interactions, making up 72% of expressed miRs, while less than 1/3 of expressed miRs (112/397) remained unaffected by treatment, treatment duration and their interactions. Of the significantly affected miRs, approximately 50% (149/285) were significant for an interaction effect between zinc depletion and duration of zinc depletion. The number of miRs significant for zinc depletion only, duration of zinc depletion only or both zinc depletion and duration was 56, 48 and 32, respectively. 2.2.3. Zinc depletion altered abundance of miR-132-3p, miR-1246, miR-1273g-3p, miR-4484, miR-4521 and miR-4787-5p Expression of selected miRs was validated after 3, 12 and 24 h TPEN treatment using qRT-PCR. The miRs investigated were miR-132-3p, miR-1246, miR-1273g-3p, miR-4484, miR-4521 and miR-4787-5p. MiR-132-3p was found to be the most responsive to zinc depletion, out of the six miRs examined by qRT-PCR. After just 3 h of TPEN treatment, the abundance of miR-132-3p was significantly increased (2.2 folds), and further increased by 17.7 and 28.5 folds after 12 and 24 h, respectively, compared to their respective control groups (Figure ). TPEN treatment resulted in a time-dependent increase in the abundance of miR-132-3p while its abundance remained the same in the control groups at 3, 12 and 24 h. Another miR substantially affected by TPEN treatment was miR-1246, which was significantly increased in a time-dependent manner. At 3 h the abundance of miR-1246 was the same between TPEN and control groups. However, TPEN treatment increased the abundance of miR-1246 by 3.8 and 18.4 folds at 12 and 24 h, respectively, compared to their  58 respective control group (Figure ). In contrast, in the control groups the abundance of miR-1246 was significantly decreased by 1.8 and 1.7 folds at 12 and 24 h, respectively, compared to that at 3 h.  Zinc depletion significantly increased the abundance of miR-4484 in a time-dependent manner (Figure ). While miR-4484 was significantly elevated at all 3 time points investigated, the peak increase in abundance occurred at 12 h TPEN treatment, with a 5.5 fold increase compared to the 12 h control group. At 3 h TPEN treatment miR-4484 abundance increased by 1.4 fold and, at 24 h TPEN treatment, by 3.5 fold compared to the respective control groups. Abundance of miR-448 in the control groups was also increased over time. The abundance of miR-4787-5p was significantly elevated by 1.6 and 4.0 folds at 3 and 24 h TPEN treatment, respectively, compared to their respective control groups, but the abundance of miR-4787-5p was unaffected by TPEN treatment at 12 h (Figure ). The abundance of miR-4787-5p remained the same from 3 to 12 h of TPEN treatment; but was increased by 3.6 folds after 24 h of TPEN treatment compared to the 3 h TPEN treatment durations. The abundance of miR-4787-5p in the control group varied, with 1.6 and 1.4 fold increases at 12 and 24 h, respectively, compared to that at 3 h. The abundance of miR-1273g-3p was decreased by 2.4 folds in the 3-h TPEN-treated group compared to the 3-h control, but was increased by 2.2 and 3.4 folds after 12 and 24 h of TPEN treatment, respectively, compared to their respective controls (Figure ). TPEN treatment significantly increased in the abundance of miR-1273g-3p in a time-dependent manner. In contrast, the abundance of miR-1273g-3p was decreased by 2.9 and 3.2 folds in  59 the 12 and 24-h control groups, respectively compared to the 3-h control group. Therefore, miR-1273 exhibited opposite patterns with decreased abundance over time in the control groups and increased abundance in the TPEN treated groups.  Zinc depletion resulted in 1.4 and 1.3 fold increases in the abundance of miR-4521 in the 3 and 12-h TPEN groups compared to their respective control groups, respectively; Figure ). However, after 24 h TPEN treatment, abundance of miR-4521 decreased by 5.1 folds compared to its control group.  Treatment duration-dependent decrease in miR-4521 abundance was observed in both the control and TPEN treated groups, but to a larger extent in the TPEN treated groups. In the control groups, miR-4521 abundance decreased by 1.4 and 2.0 fold at 12 and 24 h, respectively, compared to the 3 h control group. The 12 and 24 h TPEN treatments decreased miR-4521 by 1.6 and 14.4 folds, respectively, compared to the 3 h control group. In summary, we observed that the longer TPEN treatment duration (24 h) typically led to the largest changes in the abundance of miRs; however, the abundance of miR-4484 was observed at the highest level after 12 h TPEN treatment. Abundance of miR-132-3p, miR-1246, miR-4484, miR-4787-5p and miR-1273g-3p were significantly increased by 24 h TPEN treatment compared to their respective controls. Of the miRs assessed, the largest increase in abundance was observed for miR-132-3p, with a 28 folds increase in the 24h TPEN group, followed by miR-1246, which was increased by 18 folds in the 24 h TPEN group. MiR-4521 was the only miR among the 6 miRs assessed which decreased in abundance after 24 h TPEN treatment.  60 2.3. Discussion Zinc depletion was shown to induce apoptosis in a time-dependent manner in MDA-MB-231 breast cancer cells, similarly to previous findings from our laboratory13,238. The apoptotic effects of reduced zinc from TPEN treatment have been demonstrated in many other cell lines including thymocytes135, lymphocytes136,137,  malignant airway epithelial cells138,  leukemia cells139, retinal pigment epithelial cells140, melanoma cells141, keratinocytes142, pancreatic cancer cells143 and cervical cancer cells132. Dietary zinc deficiency also induces apoptosis in vivo in rats in multiple tissues123. While much progress has been made towards elucidating the mechanisms of zinc depletion-induced apoptosis, particularly on the central role of the intrinsic mitochondrial apoptotic pathway13,14,137,148,149, the molecular pathways involved are still poorly understood. This research provided novel information on miR expression during apoptosis arising from zinc depletion. The effect of zinc depletion on the miR expression profile of MDA-MB-231 cells was measured after just 3, 12 or 24 h of TPEN treatment (10 ??M), while the majority of cells still had intact DNA, indicating that they had not yet reached late-stage apoptosis. Expression of numerous miRs was altered after 12 and 24 h TPEN treatment, indicating possible involvement of these miRs in mediating zinc depletion-induced apoptosis. Overall, the majority of differentially expressed miRs were upregulated by zinc depletion.  Cellular zinc status may regulate post-transcriptional miR processing, as treatment of MCF-7 breast cancer cells with a chemotherapeutic agent (Clioquinol) which raised intracellular zinc to cytotoxic levels suppressed expression of Dicer as well as Ago2, and was also associated with global downregulation of miR, presumably through decreased miR  61 biogenesis and stability21. It is conceivable that in our study zinc depletion altered the miR profile by modulating miR biogenesis; however, this hypothesis requires validation with further research. The earliest miR that was significantly affected by zinc depletion was miR-182-5p, with a modest 1.3 fold decrease in its expression after just 3 h of zinc depletion. MiR-182-5p has been reported as upregulated in breast cancer, as well as in many other types of cancer, including colorectal, prostate and lung cancer239. Additionally, a recent study found that expression of miR-182-5p was significantly higher in triple negative breast cancers as well as in triple negative MDA-MB-231 breast cancer cells compared to normal breast tissue240. MiR-182-5p has been reported to act as an oncogene in a number of studies, including reported anti-apoptotic roles239. For example, inhibition of miR-182-5p expression in MDA-MB-231 cells inhibited cellular proliferation and invasion, and induced apoptosis240. In MCF-7 breast cancer cells, miR-182-5p negatively regulated FOXO1, a pro-apoptotic transcription factor, to increase cell viability241. MiR-182-5p targets include other pro-apoptotic genes including p27(Kip1) in MDA-MB-231 and HEK-293 cells, as well as the Bcl-2 family members BAK and BAX in HEK-293T cells242. Interestingly, a role for miR-182 in the regulation of zinc homeostasis has also been reported. In PrE cells, overexpression of miR-182 with a pre-miR-182 mimic negatively regulated Zip1, which transports zinc into the cytoplasm from the extracellular space or organelles, through 2 binding sites ?in ?its ?3?UTR ?resulting ?in ?Zip1 mRNA degradation227. Additionally, miR-182 upregulation decreased mRNA of 5 other zinc transporters (Zip3, Zip7, Zip9, ZnT1 and ZnT7). The overall effect of miR-182 overexpression on zinc  62 homeostasis was to lower intracellular zinc in PrE, possibly through reduced cellular zinc uptake as observed in HEK-293 cells.  Based on these findings, the decrease in miR-182-5p expression that we observed after 3 h of zinc depletion may have served to increase zinc uptake in response to intracellular zinc depletion. However, the miR microarray indicated that miR-182-5p was poorly expressed in the control group (average signal intensity: 465.1) and the 3 h TPEN treatment only induced a modest decrease in overall expression (average signal intensity: 356.2 TPEN, 1.3 fold decrease). Furthermore, no significant changes in miR-182-5p expression were observed after 12 or 24 h TPEN treatment. Due to its role in zinc homeostasis and apoptosis, miR-182-5p could possibly provide an early connection between zinc depletion and subsequent induction of apoptosis; however, more research is required to validate its expression as well as functional significance in zinc depletion-induced apoptosis in MDA-MB-231 cells.  The expression of miR-132-3p, -1246, -1273g-3p, -4484, -4521 and -4787-5p were shown to be substantially altered by zinc depletion using the microarray profiling assay and these observations were validated using qRT-PCR. Of these validated miRs, miR-132-3p displayed the greatest induction following zinc depletion. The abundance of miR-132-3p doubled after just 3 h of TPEN treatment and continued to increase to 28 times of the control group after 24 h of TPEN treatment.  Decreased expression of miR-132-3p has been previously reported in cancer and it has been found to have tumor suppressive roles219,243. For example, miR-132-3p was reported as significantly downregulated in breast DCIS, an early stage of breast cancer, compared to  63 paired samples of adjacent normal breast tissue219. Transfection of MCF-7, MDA-MB-231 and BT-549 breast cancer cell lines with pre-miR-132 mimics decreased cell viability in all three cell lines 96 h post-transfection. Furthermore, transfection of MDA-MB-231 and MCF-7 cells with miR-132 mimics for 24 h inhibited colony formation after 2-weeks, indicating that miR-132 negatively regulated anchorage-independent growth. Therefore, miR-132 appears to exert an inhibitory role in the survival of breast cancer cells.  In prostate cancer samples, expression of miR-132-3p was also significantly downregulated due to DNA methylation243. Decreased miR-132-3p expression correlated with metastasis and lymph node invasion as well as an increased likelihood of tumor recurrence. Transfection of PC-3 prostate cancer cells with a pre-miR-132 mimic caused cells to develop a rounded phenotype and become unattached from the plates at 72 h, followed by increased apoptosis at 96 h. Additionally, miR-132 upregulation dramatically reduced cellular migration and invasion of PC-3 cells. MiR-132 was found to negatively regulate talin 2, a protein that plays a role in the assembly of actin filaments, and inhibition of talin 2 was linked to decreased cellular adherence and migration.  MiR-132-3p has previously been reported to play an important role in response to nutritional stress. In response to serum deprivation, miR-132-3p was shown to rapidly increase in human preadipocytes and in vitro differentiated adipocytes244. Furthermore, miR-132 overexpression directly repressed silent information regulator 1 (SirT1) expression in preadipocytes. SirT1 inhibits activation of nuclear-factor-?B (NF-?B) by deacetylation. As a result, miR-132 upregulation inhibited SirT1-mediated deacetylation of NF-?B, ultimately resulting in NF-?B activation and translocation from the cytosol to the nucleus. Interestingly,  64 activation of NF-?B has also been reported to mediate zinc depletion-induced apoptosis in leukemia cells154. In this study it is unknown whether induction of miR-132-3p following nutritional stress was due in part to zinc depletion or solely to other nutritional deficiencies.  In accordance with these findings, our research study also found that zinc depletion, a form of nutritional stress, induced miR-132-3p expression. Since miR-132-3p was reported to have pro-apoptotic effects in prostate cancer cells243 and to decrease viability in breast cancer cells219, it may potentially serve a critical link between zinc depletion and induction of apoptosis; however, further research is required to affirm its role in zinc depletion-induced apoptosis in MDA-MB-231 breast cancer cells.  Abundance of miR-1246 was also largely elevated after 12 and 24 h of TPEN treatment. The mechanisms by which zinc depletion altered miR-1246 expression in our study are currently unknown. Reduced abundance of miR-1246 in malignant cells may occur as a result of increased cellular export. Malignant breast cancer cell lines (e.g. MDA-MB-231, MCF-7, Sk-Br3) selectively released miR-1246 into exosomes, while human breast epithelial MCF-10A cells and human lung fibroblasts IMR90 cells retained a higher amount of miR-1246245.  MiR-1246 has been reported to exert a pro-apoptotic effect. In human lung carcinoma A-549 cells, overexpression of a pre-miR-1246 mimic promoted apoptosis246. Further investigation revealed that miR-1246 negatively regulated the amount of protein kinase DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) in human lung cancer H1299 cells and human osteosarcoma U2OS cells, and was shown to directly target the ?3?UTR ?derived ?from ?DYRK1A mRNA in human colon cancer HCT-116 cells246.  65 DYRK1A is an anti-apoptotic protein that has been found to negatively regulate caspase-9 in retina cells247.  These observations collectively suggest that miR-1246 plays a role in apoptosis.  Therefore, the increased abundance of miR-1246 observed in this research may have been involved in initiating zinc depletion-induced apoptosis in MDA-MB-231 cells; however, more research is needed to verify its role in this model system.  The microarray results also indicated that zinc depletion resulted in downregulation of miR-181b-5p by greater than 2 fold after 24 hours of treatment (Table 2.3). Overexpression of miR-181b-5p occurs frequently in human breast cancer, and its expression levels are correlated with increased breast tumor aggressiveness and shorter disease free survival248?251. Increased expression of miR-181b may be induced by a variety of oncogenic pathways implicated in breast tumorigenesis involving hypoxia252 as well as transforming growth factor ??253,254, signal transducer and activation of transcription 3255, and high mobility group A1 proteins250.   Expression of miR-181b has been shown to play an important role during tumorigenesis, as inhibition of miR-181b-1 (precursor for miR-181b-5p) reduced tumor colony formation in multiple cancer cell lines including breast cancer cells (transformed MCF-10A ER-Src) as well as colon (HT-29, HCT-116), lung (A-549) and liver (Hep-3B) cancer cells255. Treatment of transformed MCF-10A ER-Src cells with anti-miR-181b-1 also inhibited cellular invasion. Furthermore, anti-miR-181b-1 inhibited tumor growth in vivo in multiple types of mouse xenografts (derived from MCF-10A ER-Src, HT-29 and HCT-116 cells). Increased expression ( > 100 fold increase) of miR-181b by transfection with pre-miR- 66 181b in MDA-MB-435s breast cancer cells promoted cell proliferation and reduced apoptosis250. Additionally, miR-181b-5p has been found to regulate tamoxifen resistance in mice. Treatment of mice with tamoxifen as well as anti-miR-181b-5p, reduced tumor growth of tamoxifen-resistant xenografts (derived from MCF-7 cells), compared to just treating with tamoxifen and a negative control256. In another study, miR-181b-5p was identified as inhibiting the DNA damage response in breast cancer, by negatively regulates ataxia telangiectasia mutated, which is involved in repairing double stranded DNA breaks248. These studies suggest that downregulation of oncogenic miR-181b-5p could play a role in regulating cellular growth and survival in response to zinc depletion, however, more research is needed. Many of the miRs identified as significantly altered by zinc depletion in this research, have only been recently discovered and their biological functions are largely unknown. For example, miR-1273g-3p was discovered in 2008 by deep sequencing of human embryonic stem cells257. MiR-4484 and miR-4521 were identified in 2010 by deep sequencing of normal and malignant B-cells258. MiR-4484 was also identified as encoded in the mitochondrial genome of HEK-293 and HeLa cells in 2012259. MiR-4787-5p was identified in 2011 by sequencing of normal and tumor breast tissue260. In the absence of knowledge in biological functions of these miRs, the significance of an increased abundance of these miRs in zinc depletion-induced apoptosis in MDA-MB-231 cells remains to be elucidated. In summary, zinc depletion induced apoptosis in a time-dependent manner in MDA-MB-231 cells.  Zinc depletion altered the abundance of numerous miRs in MDA-MB-231 cells, and differentially expressed miRs were mainly increased by zinc depletion. Notably,  67 the abundance of miR-132-3p and miR-1246 were substantially increased by zinc depletion.  Based on the previously demonstrated role of miR-132-3p and miR-1246 in promoting apoptosis243,246, these two miRs may be involved in mediating zinc depletion-induced apoptosis.  Furthermore, a considerable number of miRs identified as altered by zinc depletion have only been discovered in recent years and have yet unknown biological functions. An altered abundance of these miRs in zinc depletion-induced apoptosis indicates an association between these miRs and zinc depletion-induced apoptosis. Taken together, for the first time, this research provided evidence of an association between miRs and zinc depletion-induced apoptosis in human breast cancer MDA-MB-231 cells; however, their specific roles in the induction and regulation of zinc depletion-induced apoptosis in human breast cancer MDA-MB-231 cells remains to be elucidated.    68  Table 2.1: Differential expression of miR-182-5p induced by 3-h TPEN treatment.  MDA-MB-231 ?cells ?were ?treated ?with ?DMSO ?(control) ?or ?TPEN ?(10 ??M ?in ?DMSO) ?for ?3 ?h (n=3). The values are mean ? SD. Cutoff criteria was set at signal intensity > 500 for at least one individual sample and p < 0.05.  MiR Fold Change p-value hsa-miR-182-5p -1.3 ? 1.1E-01 4.7E-02     69  Table 2.2: Differential expression of miRs induced by 12-h TPEN treatment.   MDA-MB-231 ?cells ?were ?treated ?with ?DMSO ?(control) ?or ?TPEN ?(10 ??M ?in ?DMSO) ?for ?12 ?h ?(n=3). The values are mean ? SD. Cutoff criteria was set at magnitude fold change > 2; signal intensity > 500 for at least one individual sample, and p < 0.05.  MiR Fold Change p-value hsa-miR-3127-5p 374.1 ? 3.1E+02 6.7E-03 hsa-miR-5194 113.2 ? 6.8E+01 1.2E-03 hsa-miR-4485 34.5 ? 1.5E+01 8.4E-04 hsa-miR-132-3p 16.4 ? 2.9E+00 1.4E-03 hsa-miR-4734 3.3 ? 1.8E+00 2.8E-02 hsa-miR-1273g-3p 3.2 ? 1.3E+00 4.4E-02 hsa-miR-5096 2.5 ? 8.7E-01 1.7E-02 hsa-miR-4484 2.5 ? 5.5E-01 5.2E-03 hsa-miR-1973 2.2 ? 7.1E-01 5.0E-02 hsa-miR-4690-5p 2.2 ? 6.3E-01 1.5E-02     70  Table 2.3: Differential expression of miRs induced by 24-h TPEN treatment.   MDA-MB-231 ?cells ?were ?treated ?with ?DMSO ?(control) ?or ?TPEN ?(10 ??M ?in ?DMSO) ?for ?24 h (n=3). The values are mean ? SD. Cutoff criteria was set at magnitude fold change > 2; signal intensity > 500 for at least one individual sample, and p < 0.05.  MiR Fold Change p-value hsa-miR-30c-1-3p 15.5 ? 2.2E+00 5.7E-05 hsa-miR-132-3p 14.0 ? 5.1E+00 6.4E-03 hsa-miR-4484 11.3 ? 1.7E+00 1.4E-03 hsa-miR-4485 10.8 ? 8.6E+00 4.2E-02 hsa-miR-1246 5.5 ? 2.1E+00 2.4E-02 hsa-miR-4530 5.4 ? 2.6E+00 8.1E-03 hsa-miR-6126 5.1 ? 1.4E+00 2.1E-03 hsa-miR-4787-5p 4.5 ? 6.6E-01 4.2E-04 hsa-miR-1273g-3p 3.7 ? 1.3E+00 1.0E-02 hsa-miR-3196 3.4 ? 9.8E-01 5.7E-03 hsa-miR-4516 3.3 ? 9.9E-01 6.5E-03 hsa-miR-3960 3.1 ? 1.0E+00 2.3E-02 hsa-miR-638 3.1 ? 3.0E-01 2.5E-04 hsa-miR-4454 3.0 ? 1.3E+00 4.3E-02 hsa-miR-6125 2.9 ? 7.3E-01 5.2E-03 hsa-miR-3665 2.8 ? 6.1E-01 3.8E-03 hsa-miR-1234-5p 2.8 ? 7.7E-01 2.5E-02 hsa-miR-1973 2.7 ? 8.1E-01 2.7E-02 hsa-miR-6087 2.7 ? 5.3E-01 1.3E-02 hsa-miR-4508 2.6 ? 1.2E+00 3.5E-02 hsa-miR-1260b 2.2 ? 8.5E-01 3.9E-02 hsa-miR-4497 2.1 ? 5.4E-01 1.4E-02 hsa-miR-181b-5p -2.2 ? 1.1E-01 3.4E-02 hsa-miR-4521 -10.6 ? 3.6E-02 1.9E-03   71     Figure 2.1: Zinc depletion-induced apoptosis in MDA-MB-231 cells.  MDA-MB-231 ?cells ?were ?treated ?with ?DMSO ?only ?(control) ?or ?TPEN ?(10 ??M ?in ?DMSO) for 0, 3, 6, 12, 24, 48 or 72 h. Apoptosis was assessed by PI-staining flow cytometry.  3 6 12 24 48 720102030Treatment duration (h)DNA fragmentation (%) ControlTPEN 72      Figure 2.2: 3-h TPEN treatment altered expression of several miRs. MDA-MB-231 cells were treated with DMSO (control) ?or ?TPEN ?(10 ??M ?in ?DMSO) ?for ?3 ?h. ?Red indicates upregulation, black indicates no change and green indicates downregulation (p < 0.05).  73  Figure 2.3: 12-h TPEN treatment altered expression of many miRs.  MDA-MB-231 ?cells ?were ?treated ?with ?DMSO ? ?(control) ?or ?TPEN ?(10 ??M ?in ?DMSO) ?for ?12 ?h. Red indicates upregulation, black indicates no change and green indicates downregulation (p<0.05).   74    Figure 2.4: 24-h TPEN treatment altered expression of many miRs.  MDA-MB-231 ?cells ?were ?treated ?with ?DMSO ? ?(control) ?or ?TPEN ?(10 ??M ?in ?DMSO) ?for ?24 ?h. Red indicates upregulation, black indicates no change and green indicates downregulation (p<0.05).   75    Figure 2.5: Zinc depletion promoted a time-dependent increase in hsa-miR-132-3p expression. MDA-MB-231 cells were treated with DMSO  (control) or TPEN TPEN (10 ??M ?in ?DMSO) ?for 3, 12 or 24 h. Values are mean ? SD (n = 6). Expression is reported as relative to the control at 3 h. Lower case letters indicate the significance among the control groups while upper case letters indicate the significance among the TPEN treated groups. Different letters indicate significant differences among the means within the same treatment group. Asterisks indicate significant differences between the control and TPEN-treated groups at the same time point (p < 0.05). 3 12 24 010203040Treatment duration (h)Relative abundance of hsa-miR-132-3p (fold change) a A a aBCControl TPEN *** 76   Figure 2.6: Zinc depletion increased expression of hsa-miR-1246 after 12 and 24 h TPEN treatment. MDA-MB-231 cells were treated with control (DMSO) or TPEN (10 ?M in DMSO) for 3, 12 and 24 h. Values are mean ? SD (n = 6 except TPEN at 12 h where n = 5 due to removal of an outlier).  Expression is reported relative to the control sample at 3 h. Lower case letters indicate the control groups while upper case letters indicate the TPEN treated groups. Different letters indicate significant differences between means within a treatment group. Asterisks indicate significant differences between control and TPEN-treated groups at a single time point (n=6, except 12h TPEN group, where n=5 due to removal of an outlier (p < 0.05). 3 12 24 051015Treatment duration (h)Relative abundance of hsa-miR-1246 (fold change) a A b bBCControl TPEN ** 77   Figure 2.7: Zinc depletion-induced time-dependent upregulation of hsa-miR-4484.  MDA-MB-231 cells were treated with control (DMSO) or TPEN (10 ?M ?in ?DMSO) ?for ?3, ?12 and 24 h. Values are mean ? SD (n = 6 except TPEN at 3 h and Control at 24 h, where n = 5 due to removal of outliers). Expression is reported relative to the control sample at 3 h. Lower case letters indicate the control groups while upper case letters indicate the TPEN treated groups. Different letters indicate significant differences between means within a treatment group. Asterisks indicate significant differences between control and TPEN-treated groups at a single time point (p < 0.05). 3 12 24 0246810Treatment duration (h)Relative abundance of hsa-miR-4484 (fold change) a A bcBCControl TPEN *** 78   Figure 2.8: Zinc depletion increased hsa-miR-4787-5p expression at 3 and 12 h.  MDA-MB-231 cells were treated with control (DMSO) or TPEN (10 ?M in DMSO) for 3, 12 and 24 h. Values are mean ? SD (n = 6 except TPEN at 3 h and 12 h, where n = 5 due to removal of outliers). Expression is reported relative to the control sample at 3 h. Lower case letters indicate the control groups while upper case letters indicate the TPEN treated groups. Different letters indicate significant differences between means within a treatment group. Asterisks indicate significant differences between control and TPEN-treated groups at a single time point (p < 0.05). 3 12 24 02468Treatment duration (h)Relative abundance of hsa-miR-4787-5p (fold change) aA b bAB Control TPEN ** 79   Figure 2.9: Zinc depletion promoted expression of a-miR-1273g-3p in a time-dependent manner.  MDA-MB-231 cells were treated with control (DMSO) or TPEN (10 ?M in DMSO) for 3, 12 and 24 h. Values are mean ? SD (n = 6 except TPEN at 24 h, where n = 5 due to removal of an outlier). Expression is reported relative to the control sample at 3 h. Lower case letters indicate the control groups while upper case letters indicate the TPEN treated groups. Different letters indicate significant differences between means within a treatment group. Asterisks indicate significant differences between control and TPEN-treated groups at a single time point (p < 0.05). 3 12 24 0.00.51.01.5Treatment duration (h)Relative abundance of hsa-miR-1273g-3p (fold change)aA b bBC Control TPEN *** 80   Figure 2.10: Zinc depletion inhibited has-miR-4521 expression in a time-dependent manner.  MDA-MB-231 cells were treated with control (DMSO) or TPEN (10 ?M in DMSO) for 3, 12 and 24 h. Values are mean ? SD (n = 6). Expression is reported relative to the control sample at 3 h. Lower case letters indicate the control groups while upper case letters indicate the TPEN treated groups. Different letters indicate significant differences between means within a treatment group. Asterisks indicate significant differences between control and TPEN-treated groups at a single time point (p < 0.05). 3 12 24 0.00.51.01.52.0Treatment duration (h)Relative abundance of hsa-miR-4521 (fold change) aAbcBCControl TPEN *** 81 Chapter 3: Conclusions, Limitations, and Future Directions 3.1. Conclusions The effect of zinc depletion on the human global microRNA expression profile (>2000 miRs) in human breast cancer MDA-MB-231 cells was investigated in this study. Although it has been previously shown that acute zinc deficiency altered serum miR expression profile in young human males19 and chronic zinc deficiency altered tissue miR expression profile in rats20, the effect of TPEN-induced zinc depletion on the miR expression profile in vitro was unknown and this was the first study to investigate the topic. In this study, zinc depletion altered expression of many miRs in a time-dependent manner. After just 3 h of ?zinc ?depletion ?using ?TPEN ?(10 ??M), ?the ?miR ?expression ?profile ?was largely unaffected, one exception being miR-182-5p, which was significantly downregulated. In contrast, after 12 and 24 h of zinc depletion, approximately one-quarter of the expressed miRs were differentially affected with the majority being upregulated. In total, almost 75% of the expressed miRs were significantly affected by zinc depletion, treatment-duration and their interactions, providing evidence of a broad ranging effect of zinc depletion on the miR expression profile in human breast cancer MDA-MB-231 cells.  Zinc is known to exert diverse physiological functions as it is required for growth, reproduction, neurological development, immune function, etc.1 but the molecular basis for zinc?s ?wide-ranging physiological functions are not well understood. This research provided novel evidence that cellular zinc status greatly impacted the miR expression profile. The extent of gene-regulation by miRs is broad ranging and complex; with more than 60% of  82 protein-coding genes predicted as miR targets168, and these collective miR-target interactions drive cellular functions. Evidence obtained from this study, together with published evidence, provides a foundation for a new mode of zinc-mediated cellular regulation at the level of miR expression, which could help explain the diverse effects of zinc deficiency. However, the functional significance of zinc depletion-induced changes to miR expression needs to be validated in further research studies. 3.2. Limitations There are several limitations associated with this research. One such limitation was the use on an in vitro system with a single breast cancer cell line. The MDA-MB-231 cell line employed has a high invasive potential compared to other breast cancer cell lines (e.g. non-invasive MCF-7)261. MDA-MB-231 cells are also triple negative (ER-, PR- and HER2-) and express mutated p53. MiR expression has been reported to vary between breast cancer cell lines, with decreased expression of two-thirds of miRs investigated in triple negative human breast cancer MDA-MB-231 and BT-549 cells compared to ER+ human breast cancer MCF-7 and T47D cells209. In breast tumors, the overall global miR expression level was also found to be lower in more aggressive ER- tumors201 and also decreased with higher tumor grade204. On average, breast cancer cell lines had lower global miR expression compared to breast tumors204. Reduced expression of miRs in more aggressive cancers may be due to differences in miR biogenesis and stability201,204,209. Therefore, the effects of zinc depletion might influence miR expression in a cell type specific manner. Investigation into the effects of zinc depletion-induced apoptosis on miR expression in other breast cancer cell lines and in  83 vivo (e.g. rodent experimental model) would provide a better understanding of the influence of zinc status on miR expression.  Another limitation is that the microarray profiling data showed a significant increase in the expression of miR-30c-1-3p and miR-638 in response to zinc depletion; however these effects of zinc were unable to be validated by qRT-PCR. MiR-30c-1-3p could not be validated by qRT-PCR due to extremely low expression (cycle threshold > 35 for 100 ng starting RNA, data not shown). MiR-638 also showed inconsistent expression in a dose curve study so it was not examined further. Generally, the qRT-PCR results were consistent with the results obtained using microarray profiling assay, although the magnitude of change varied somewhat. For example, microarray assay and qRT-PCR assay showed a 14.0 and 28.5 fold increase, respectively, in the expression of miR-132-3p after 24 h of TPEN treatment (Table 2.3, Figure ). The results after 12 h TPEN treatment were more consistent with the microarray assay and qRT-PCR assay indicating a 16.4 and 17.7 fold increases, respectively, in expression of miR-132-3p (Table 2.2, Figure ). Additionally, qRT-PCR assay detected a 2.2 fold increase in the abundance of miR-132-3p after 3 h of TPEN treatment, while the microarray profiling assay showed that TPEN treatment had no effect on the expression of miR-132-3p (Figure , Figure ). These discrepancies may be due to biological and experimental variations, as well as differences between these two platforms. In a study comparing miR expression as measured by TaqMan qRT-PCR-array to miRNA microarray analysis by LC Sciences (the same company that performed the miR microarray profiling assay reported in this thesis), a low correlation was observed (r=-0.443) indicating large  84 variability between the two platforms262. The variation between these two methods was inversely proportional to the level of miR expression, with the largest variation for miRs with low abundance. The sensitivity of TaqMan qRT-PCR is greater than microarray, and has a dynamic range of 7 logs232 compared to a >3.5 log dynamic range reported by LC sciences263, which may account for some of the variation observed for poorly expressed miRs. The LC Sciences microarray was also found to have a higher false positive rate between technical replicates (13%) compared to the TaqMan qPCR-array (1%). Therefore, qRT-PCR ?has ?been ?used ?as ?a ??gold ?standard? ?due ?to ?its ?high ?sensitivity ?and ?specificity234.  Despite these differences that we observed between the microarray and qRT-PCR platforms, the microarray still served as a valuable cost-effective and rapid screening tool for identifying differentially expressed miRs. The microarray analysis indicated many changes in zinc depletion-induced alterations in miR expression, of which the abundance of 6 miRs was confirmed by qRT-PCR. Overall, the qRT-PCR results tend to be in line with the microarray findings that numerous miRs were altered by zinc depletion-induced apoptosis; however, changes in expression of individual miRs should be validated using qRT-PCR.  3.3. Future Directions The microarray profiling results indicated that many miRs were significantly altered by zinc depletion and only a handful of these miRs were validated by qRT-PCR in this research. There are future opportunities to validate expression of other miRs significantly affected by zinc depletion using qRT-PCR.  85 Further research is required to establish the role of miR up/down regulation on zinc depletion-induced apoptosis in breast cancer cells. Some of the miRs identified as significantly altered by zinc depletion in our research have been implicated in apoptosis in other types of cells; however the functional significance in our model system is still unknown. For example, it would be interesting to examine the role of miR-132-3p upregulation in zinc depletion-induced apoptosis, since miR-132-3p overexpression has been associated with decreased viability of breast cancer cells including MDA-MB-231 cells219, and induction of apoptosis in PC-3 cells243. MiR-1246, which was also substantially upregulated during zinc depletion-induced apoptosis, promoted apoptosis in human lung carcinoma A549 cells246. Additionally, our work identified some miRs (miR-1273, -4484, -4521 and -4787-5p) as significantly affected by zinc depletion, but they have no reported role in apoptosis at the time of this work. Currently, work is underway in the Xu laboratory to establish stable miR transfected cell lines using a PiggyBac transposon vector system, in order to investigate the functional significance of some of these miRs in zinc depletion-induced apoptosis.  Finally, it would be interesting to examine the pathways involved in altered miR expression during zinc depletion-induced apoptosis. It has been previously reported that increased intracellular zinc at cytotoxic levels reduced expression of proteins involved in miR biogenesis and stability (Dicer and Ago2), however the mechanisms involved here are unknown14. Further research is required to assess the pathways underlying changes in miR expression during zinc depletion and whether or not this involves changes to miR processing and stability.   86 In summary, this work provides novel evidence that zinc depletion regulates miR expression, opening the door for research opportunities into the mechanisms of zinc depletion-induced alterations in miR expression as well as the functional significance of zinc-regulated miRs.   87 References 1. Maret, W. & Sandstead, H. H. Zinc requirements and the risks and benefits of zinc supplementation. J. Trace. Elem. Med. Biol. 20, 3?18 (2006). 2. Alam, S. & Kelleher, S. L. Cellular mechanisms of zinc dysregulation: a perspective on zinc homeostasis as an etiological factor in the development and progression of breast cancer. Nutrients 4, 875?903 (2012). 3. Manning, D. L. et al. Oestrogen-regulated genes in breast cancer: association of pLIV1 with lymph node involvement. Eur. J. Cancer 30A, 675?678 (1994). 4. 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MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-let-7a-2-3p 29 ? 4 30 ? 4 27 ? 7 14 ? 7 28 ? 9 9 ? 6 hsa-let-7a-3p 16 ? 6 20 ? 2 29 ? 4 10 ? 2 33 ? 23 32 ? 8 hsa-let-7a-5p 12,488 ? 1,406 11,499 ? 550 14,485 ? 4,324 14,231 ? 1,978 10,416 ? 1,458 11,937 ? 2,151 hsa-let-7b-3p 14 ? 2 16 ? 3 24 ? 8 14 ? 3 24 ? 6 15 ? 5 hsa-let-7b-5p 2,509 ? 319 2,446 ? 964 3,802 ? 2,168 2,461 ? 2,107 1,350 ? 328 2,909 ? 1,806 hsa-let-7c 8,789 ? 757 7,963 ? 826 10,001 ? 3,477 8,075 ? 3,189 5,635 ? 1,597 6,713 ? 2,035 hsa-let-7d-3p 57 ? 8 71 ? 13 86 ? 20 81 ? 11 72 ? 23 41 ? 20 hsa-let-7d-5p 10,046 ? 919 9,678 ? 720 11,610 ? 3,473 11,658 ? 982 8,363 ? 1,606 9,189 ? 690 hsa-let-7e-5p 8,682 ? 622 7,067 ? 1,495 10,290 ? 3,616 8,586 ? 2,885 5,438 ? 1,470 6,171 ? 2,472 hsa-let-7f-1-3p 21 ? 6 22 ? 2 28 ? 6 12 ? 3 23 ? 5 14 ? 6 hsa-let-7f-5p 11,794 ? 2,631 11,942 ? 547 14,362 ? 3,580 14,663 ? 1,605 11,409 ? 1,441 12,250 ? 1,545 hsa-let-7g-5p 3,495 ? 226 3,209 ? 106 3,847 ? 1,035 3,188 ? 838 2,911 ? 342 3,135 ? 923 hsa-let-7i-5p 3,063 ? 158 2,781 ? 125 2,611 ? 452 3,218 ? 535 2,848 ? 543 2,808 ? 164 hsa-miR-7-1-3p 63 ? 9 52 ? 6 65 ? 14 30 ? 7 60 ? 7 30 ? 12 hsa-miR-7-5p 1,082 ? 295 1,016 ? 118 1,435 ? 197 1,127 ? 81 788 ? 104 831 ? 144 hsa-miR-10a-5p 189 ? 32 185 ? 23 177 ? 10 120 ? 11 157 ? 26 182 ? 38 hsa-miR-10b-5p 119 ? 36 131 ? 28 136 ? 16 75 ? 15 109 ? 24 79 ? 22 hsa-miR-15a-5p 323 ? 40 326 ? 2 318 ? 62 235 ? 32 463 ? 45 211 ? 104 hsa-miR-15b-3p 45 ? 7 23 ? 11 46 ? 10 20 ? 3 35 ? 11 23 ? 3 hsa-miR-15b-5p 3,942 ? 535 3,717 ? 659 3,341 ? 765 3,209 ? 454 5,753 ? 629 2,816 ? 1,213 hsa-miR-16-2-3p 33 ? 12 36 ? 8 47 ? 4 25 ? 4 38 ? 14 25 ? 7 hsa-miR-16-5p 5,332 ? 1,526 5,272 ? 326 4,392 ? 875 5,035 ? 475 6,085 ? 640 4,507 ? 1,053 hsa-miR-17-5p 1,011 ? 75 950 ? 127 1,245 ? 121 929 ? 106 778 ? 150 512 ? 60 hsa-miR-18a-5p 40 ? 8 38 ? 3 42 ? 8 31 ? 6 45 ? 13 30 ? 20  108 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-18b-5p 18 ? 4 16 ? 6 23 ? 1 12 ? 2 25 ? 10 14 ? 12 hsa-miR-19a-3p 36 ? 18 43 ? 9 47 ? 10 26 ? 3 43 ? 8 21 ? 8 hsa-miR-19b-3p 426 ? 107 690 ? 346 276 ? 61 382 ? 87 190 ? 24 123 ? 54 hsa-miR-20a-5p 1,138 ? 369 1,053 ? 159 1,396 ? 78 1,060 ? 44 949 ? 210 562 ? 65 hsa-miR-20b-5p 391 ? 44 457 ? 32 639 ? 149 373 ? 188 257 ? 51 218 ? 118 hsa-miR-21-5p 9,473 ? 1,659 9,190 ? 619 10,598 ? 693 8,626 ? 827 11,333 ? 1,764 10,488 ? 1,501 hsa-miR-22-3p 746 ? 33 753 ? 17 645 ? 115 585 ? 77 809 ? 147 580 ? 229 hsa-miR-22-5p 55 ? 19 92 ? 32 84 ? 7 68 ? 14 98 ? 13 67 ? 29 hsa-miR-23a-3p 6,494 ? 1,374 6,638 ? 471 7,032 ? 246 6,147 ? 379 6,966 ? 216 6,888 ? 505 hsa-miR-23b-3p 6,762 ? 507 7,315 ? 273 6,585 ? 510 6,308 ? 235 6,859 ? 648 6,696 ? 214 hsa-miR-23c 1,623 ? 332 1,663 ? 454 1,813 ? 32 1,249 ? 282 1,165 ? 486 1,351 ? 590 hsa-miR-24-1-5p 14 ? 17 1 ? 1 3 ? 0 1 ? 1 4 ? 2 2 ? 1 hsa-miR-24-2-5p 30 ? 8 28 ? 3 34 ? 10 17 ? 6 47 ? 15 19 ? 12 hsa-miR-24-3p 4,868 ? 236 5,203 ? 465 4,349 ? 300 4,319 ? 374 4,801 ? 634 4,517 ? 780 hsa-miR-25-3p 1,184 ? 34 1,103 ? 313 1,416 ? 167 1,121 ? 284 1,070 ? 92 1,077 ? 50 hsa-miR-26a-5p 6,129 ? 850 5,594 ? 568 5,246 ? 164 4,402 ? 353 6,703 ? 465 3,967 ? 501 hsa-miR-26b-5p 1,358 ? 116 1,104 ? 261 1,671 ? 365 979 ? 399 1,154 ? 246 1,270 ? 502 hsa-miR-27a-3p 1,328 ? 57 1,457 ? 156 1,315 ? 179 1,029 ? 50 1,456 ? 32 1,012 ? 107 hsa-miR-27b-3p 815 ? 153 793 ? 62 721 ? 73 547 ? 60 1,094 ? 94 625 ? 116 hsa-miR-28-3p 94 ? 29 90 ? 6 112 ? 9 95 ? 22 107 ? 3 73 ? 42 hsa-miR-28-5p 176 ? 33 186 ? 11 146 ? 8 122 ? 29 137 ? 44 136 ? 34 hsa-miR-29a-3p 2,734 ? 1,056 3,072 ? 232 3,312 ? 639 3,056 ? 448 2,963 ? 192 3,144 ? 376 hsa-miR-29a-5p 44 ? 6 37 ? 4 31 ? 6 25 ? 9 28 ? 13 13 ? 1 hsa-miR-29b-1-5p 38 ? 1 19 ? 9 37 ? 13 18 ? 10 27 ? 11 5 ? 4 hsa-miR-29b-3p 3,071 ? 552 2,710 ? 392 3,650 ? 410 2,426 ? 376 3,178 ? 347 1,701 ? 511 hsa-miR-29c-3p 549 ? 150 603 ? 15 818 ? 186 444 ? 390 309 ? 125 1,040 ? 811 hsa-miR-30a-3p 632 ? 87 660 ? 92 593 ? 72 521 ? 16 548 ? 51 386 ? 52  109 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-30a-5p 8,651 ? 831 8,699 ? 824 7,119 ? 1,125 9,065 ? 794 8,994 ? 894 7,488 ? 1,614 hsa-miR-30b-3p 23 ? 5 26 ? 0 33 ? 7 26 ? 4 48 ? 12 61 ? 10 hsa-miR-30b-5p 8,888 ? 808 8,423 ? 1,091 7,992 ? 1,045 8,639 ? 2,040 8,573 ? 607 8,954 ? 1,887 hsa-miR-30c-1-3p 37 ? 5 47 ? 5 42 ? 5 86 ? 4 84 ? 9 1,307 ? 117 hsa-miR-30c-2-3p 19 ? 4 25 ? 4 20 ? 1 14 ? 1 24 ? 2 72 ? 30 hsa-miR-30c-5p 9,767 ? 2,202 9,799 ? 178 8,408 ? 1,539 10,549 ? 599 10,691 ? 944 8,909 ? 464 hsa-miR-30d-5p 5,773 ? 571 6,226 ? 365 4,750 ? 637 5,655 ? 443 5,441 ? 678 4,152 ? 549 hsa-miR-30e-3p 437 ? 133 468 ? 39 479 ? 47 322 ? 103 369 ? 62 293 ? 51 hsa-miR-30e-5p 6,652 ? 1,489 7,371 ? 549 5,872 ? 946 6,213 ? 689 6,566 ? 236 5,715 ? 253 hsa-miR-32-3p 57 ? 5 45 ? 10 85 ? 18 29 ? 6 52 ? 3 23 ? 10 hsa-miR-34b-3p 22 ? 9 22 ? 1 27 ? 5 20 ? 3 36 ? 10 13 ? 7 hsa-miR-34c-3p 76 ? 14 90 ? 5 67 ? 15 50 ? 2 84 ? 16 41 ? 16 hsa-miR-34c-5p 32 ? 5 27 ? 3 26 ? 3 15 ? 7 37 ? 8 14 ? 9 hsa-miR-92a-3p 1,357 ? 242 1,593 ? 244 1,552 ? 112 1,639 ? 360 1,499 ? 504 975 ? 324 hsa-miR-92b-3p 755 ? 35 708 ? 119 635 ? 79 659 ? 187 730 ? 166 311 ? 136 hsa-miR-93-5p 392 ? 160 415 ? 17 463 ? 51 417 ? 72 438 ? 52 333 ? 65 hsa-miR-96-5p 55 ? 18 66 ? 11 76 ? 16 39 ? 10 101 ? 10 44 ? 6 hsa-miR-98-5p 2,123 ? 314 2,547 ? 229 3,871 ? 2,149 2,624 ? 1,923 1,368 ? 383 3,151 ? 1,874 hsa-miR-99a-5p 3,495 ? 956 4,207 ? 640 4,126 ? 392 3,080 ? 1,027 3,058 ? 837 3,692 ? 1,674 hsa-miR-99b-5p 921 ? 38 614 ? 449 652 ? 149 683 ? 222 1,002 ? 190 550 ? 178 hsa-miR-100-3p 76 ? 26 68 ? 31 82 ? 22 13 ? 5 94 ? 18 11 ? 8 hsa-miR-100-5p 8,821 ? 925 8,692 ? 615 8,458 ? 969 8,987 ? 1,264 7,178 ? 1,081 8,565 ? 1,021 hsa-miR-101-3p 5 ? 3 8 ? 1 8 ? 1 5 ? 5 19 ? 14 6 ? 3 hsa-miR-103a-3p 2,081 ? 185 2,068 ? 81 1,863 ? 118 1,993 ? 225 1,945 ? 495 1,193 ? 351 hsa-miR-106a-5p 874 ? 363 892 ? 163 1,203 ? 57 910 ? 44 755 ? 94 481 ? 39 hsa-miR-106b-3p 64 ? 5 67 ? 1 57 ? 11 58 ? 4 67 ? 4 34 ? 9 hsa-miR-106b-5p 667 ? 71 559 ? 272 568 ? 14 510 ? 95 473 ? 164 206 ? 73  110 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-107 1,901 ? 129 1,972 ? 9 1,887 ? 181 1,923 ? 202 1,965 ? 479 1,230 ? 356 hsa-miR-122-5p 20 ? 2 20 ? 1 7 ? 2 29 ? 15 9 ? 9 34 ? 14 hsa-miR-125a-5p 1,875 ? 645 2,101 ? 223 2,078 ? 47 1,877 ? 64 1,823 ? 138 1,282 ? 203 hsa-miR-125b-5p 4,161 ? 359 4,420 ? 556 5,489 ? 333 4,622 ? 179 3,770 ? 268 3,833 ? 421 hsa-miR-126-3p 98 ? 19 90 ? 19 80 ? 21 54 ? 15 110 ? 9 40 ? 24 hsa-miR-128 116 ? 6 104 ? 8 86 ? 33 68 ? 29 140 ? 55 60 ? 54 hsa-miR-130a-3p 1,519 ? 278 1,346 ? 80 1,251 ? 102 1,048 ? 90 1,767 ? 348 940 ? 278 hsa-miR-130b-3p 285 ? 76 236 ? 41 278 ? 17 237 ? 22 310 ? 74 218 ? 80 hsa-miR-130b-5p 28 ? 4 28 ? 6 32 ? 2 30 ? 3 28 ? 11 20 ? 9 hsa-miR-132-3p 49 ? 7 61 ? 6 38 ? 2 630 ? 110 47 ? 4 663 ? 234 hsa-miR-132-5p 4 ? 2 7 ? 1 2 ? 1 92 ? 5 6 ? 4 109 ? 16 hsa-miR-138-1-3p 46 ? 5 38 ? 7 32 ? 4 30 ? 8 73 ? 12 30 ? 14 hsa-miR-138-5p 301 ? 118 322 ? 13 252 ? 40 253 ? 59 438 ? 79 255 ? 133 hsa-miR-139-5p 48 ? 17 54 ? 7 42 ? 7 38 ? 12 62 ? 11 24 ? 15 hsa-miR-140-3p 532 ? 73 546 ? 36 369 ? 99 575 ? 200 793 ? 162 390 ? 326 hsa-miR-146a-5p 2,196 ? 404 1,690 ? 358 1,563 ? 359 1,429 ? 305 2,010 ? 352 919 ? 413 hsa-miR-146b-5p 188 ? 84 220 ? 59 236 ? 74 118 ? 96 134 ? 31 167 ? 85 hsa-miR-148b-3p 264 ? 82 311 ? 23 251 ? 82 233 ? 38 373 ? 140 256 ? 145 hsa-miR-149-3p 47 ? 18 47 ? 10 54 ? 13 71 ? 33 41 ? 3 188 ? 25 hsa-miR-151a-3p 266 ? 36 251 ? 43 251 ? 35 240 ? 49 299 ? 68 183 ? 108 hsa-miR-151a-5p 1,970 ? 381 1,806 ? 100 1,447 ? 308 1,822 ? 214 1,915 ? 419 1,197 ? 602 hsa-miR-151b 1,940 ? 443 1,890 ? 169 1,391 ? 361 1,814 ? 271 1,891 ? 260 1,108 ? 600 hsa-miR-152 71 ? 8 68 ? 5 64 ? 15 43 ? 13 89 ? 20 32 ? 24 hsa-miR-181a-2-3p 25 ? 2 25 ? 21 25 ? 5 18 ? 8 26 ? 9 17 ? 6 hsa-miR-181a-5p 2,391 ? 1,092 2,668 ? 381 2,227 ? 564 3,001 ? 558 3,237 ? 308 2,456 ? 1,020 hsa-miR-181b-5p 1,202 ? 435 845 ? 85 1,025 ? 137 770 ? 24 867 ? 103 392 ? 86 hsa-miR-181c-5p 548 ? 230 766 ? 113 451 ? 138 419 ? 84 509 ? 237 314 ? 84  111 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-181d 565 ? 202 548 ? 168 586 ? 70 352 ? 138 393 ? 66 201 ? 83 hsa-miR-182-5p 465 ? 52 356 ? 32 532 ? 47 478 ? 20 580 ? 76 323 ? 85 hsa-miR-183-5p 177 ? 27 151 ? 17 147 ? 40 136 ? 12 169 ? 24 106 ? 62 hsa-miR-185-5p 72 ? 5 54 ? 15 73 ? 17 53 ? 6 93 ? 18 41 ? 27 hsa-miR-186-5p 266 ? 72 227 ? 71 241 ? 69 194 ? 43 313 ? 40 123 ? 74 hsa-miR-191-5p 2,583 ? 938 3,801 ? 188 2,897 ? 637 3,681 ? 494 3,803 ? 411 3,177 ? 1,430 hsa-miR-192-5p 40 ? 4 39 ? 11 38 ? 8 60 ? 7 42 ? 11 67 ? 24 hsa-miR-193a-3p 31 ? 25 35 ? 4 29 ? 8 20 ? 5 61 ? 26 27 ? 11 hsa-miR-193b-3p 32 ? 16 37 ? 8 22 ? 6 18 ? 3 43 ? 33 22 ? 5 hsa-miR-194-5p 22 ? 1 30 ? 10 27 ? 6 49 ? 8 45 ? 5 57 ? 46 hsa-miR-195-5p 146 ? 64 176 ? 26 170 ? 31 117 ? 62 131 ? 30 210 ? 133 hsa-miR-197-3p 72 ? 36 67 ? 17 81 ? 9 128 ? 24 93 ? 27 73 ? 22 hsa-miR-197-5p 20 ? 6 24 ? 13 24 ? 11 28 ? 15 10 ? 8 39 ? 26 hsa-miR-200b-3p 42 ? 12 44 ? 2 62 ? 7 25 ? 9 67 ? 8 40 ? 9 hsa-miR-200c-3p 23 ? 1 29 ? 5 35 ? 3 24 ? 1 33 ? 7 31 ? 6 hsa-miR-206 7 ? 3 6 ? 3 24 ? 10 79 ? 33 2 ? 2 11 ? 3 hsa-miR-210 59 ? 13 57 ? 4 54 ? 12 51 ? 22 113 ? 32 55 ? 38 hsa-miR-214-3p 14 ? 7 16 ? 4 14 ? 3 20 ? 13 15 ? 4 12 ? 2 hsa-miR-215 24 ? 9 28 ? 7 32 ? 2 38 ? 8 24 ? 12 46 ? 5 hsa-miR-221-3p 10,377 ? 1,583 8,811 ? 1,408 9,506 ? 1,427 9,690 ? 695 9,460 ? 389 10,390 ? 549 hsa-miR-221-5p 290 ? 42 266 ? 9 291 ? 19 149 ? 25 308 ? 29 131 ? 55 hsa-miR-222-3p 8,961 ? 3,729 11,374 ? 259 11,335 ? 703 11,734 ? 869 11,496 ? 343 11,038 ? 158 hsa-miR-222-5p 100 ? 25 84 ? 17 133 ? 18 12 ? 4 67 ? 20 13 ? 8 hsa-miR-224-5p 245 ? 64 203 ? 61 184 ? 18 132 ? 21 221 ? 28 129 ? 30 hsa-miR-301a-3p 93 ? 28 73 ? 5 75 ? 17 39 ? 16 119 ? 30 28 ? 19 hsa-miR-320a 730 ? 93 638 ? 99 875 ? 147 939 ? 140 841 ? 73 675 ? 307 hsa-miR-320b 633 ? 157 616 ? 46 732 ? 109 864 ? 143 658 ? 88 483 ? 193  112 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-320c 649 ? 116 644 ? 13 783 ? 167 900 ? 146 664 ? 81 532 ? 210 hsa-miR-320d 559 ? 44 542 ? 66 726 ? 147 828 ? 120 624 ? 111 480 ? 177 hsa-miR-320e 601 ? 122 553 ? 33 617 ? 63 717 ? 69 560 ? 80 428 ? 139 hsa-miR-324-5p 70 ? 32 55 ? 13 66 ? 3 61 ? 14 81 ? 21 45 ? 25 hsa-miR-328 2 ? 1 4 ? 4 23 ? 10 12 ? 6 1 ? 1 2 ? 1 hsa-miR-330-3p 17 ? 3 20 ? 4 14 ? 7 9 ? 5 26 ? 10 11 ? 8 hsa-miR-331-3p 71 ? 4 78 ? 11 76 ? 14 47 ? 12 82 ? 12 38 ? 14 hsa-miR-335-3p 54 ? 7 64 ? 6 85 ? 1 30 ? 10 46 ? 21 29 ? 7 hsa-miR-335-5p 392 ? 33 365 ? 51 363 ? 86 269 ? 41 531 ? 83 238 ? 77 hsa-miR-340-5p 56 ? 9 60 ? 9 67 ? 7 38 ? 7 81 ? 5 36 ? 14 hsa-miR-342-3p 76 ? 25 95 ? 8 76 ? 14 64 ? 11 79 ? 7 82 ? 53 hsa-miR-345-5p 31 ? 5 26 ? 4 28 ? 4 13 ? 2 37 ? 2 16 ? 8 hsa-miR-346 3 ? 2 3 ? 1 19 ? 15 8 ? 6 1 ? 1 2 ? 1 hsa-miR-361-5p 291 ? 67 221 ? 8 244 ? 44 158 ? 51 363 ? 129 131 ? 68 hsa-miR-362-5p 30 ? 8 25 ? 2 23 ? 6 27 ? 11 27 ? 6 10 ? 8 hsa-miR-365a-3p 93 ? 47 141 ? 34 172 ? 18 76 ? 44 128 ? 26 103 ? 50 hsa-miR-374a-5p 129 ? 60 151 ? 25 210 ? 39 89 ? 51 147 ? 42 142 ? 84 hsa-miR-374b-5p 336 ? 37 285 ? 55 370 ? 77 231 ? 35 389 ? 42 263 ? 24 hsa-miR-374c-5p 187 ? 13 225 ? 16 248 ? 66 167 ? 5 226 ? 82 186 ? 10 hsa-miR-378a-3p 132 ? 17 129 ? 14 100 ? 28 78 ? 25 119 ? 28 58 ? 45 hsa-miR-378c 34 ? 11 29 ? 21 26 ? 15 18 ? 5 33 ? 10 17 ? 14 hsa-miR-378d 70 ? 9 70 ? 7 55 ? 10 41 ? 11 76 ? 21 37 ? 32 hsa-miR-378e 167 ? 19 159 ? 13 145 ? 21 97 ? 31 168 ? 27 79 ? 45 hsa-miR-378f 42 ? 2 46 ? 7 42 ? 9 24 ? 10 40 ? 8 21 ? 11 hsa-miR-378g 86 ? 5 74 ? 21 60 ? 13 38 ? 16 83 ? 15 35 ? 30 hsa-miR-378i 84 ? 8 72 ? 2 58 ? 8 41 ? 4 69 ? 7 33 ? 24 hsa-miR-379-5p 1 ? 1 0 ? 1 1 ? 1 26 ? 10 2 ? 1 4 ? 1  113 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-381-5p 5 ? 3 13 ? 7 3 ? 2 8 ? 6 18 ? 12 22 ? 12 hsa-miR-421 65 ? 18 46 ? 6 60 ? 6 35 ? 16 76 ? 3 36 ? 22 hsa-miR-422a 26 ? 8 26 ? 5 18 ? 6 7 ? 3 9 ? 5 12 ? 6 hsa-miR-423-5p 317 ? 28 371 ? 11 283 ? 63 281 ? 83 476 ? 98 242 ? 121 hsa-miR-424-3p 25 ? 12 18 ? 2 15 ? 3 7 ? 5 30 ? 10 7 ? 5 hsa-miR-424-5p 559 ? 47 491 ? 71 552 ? 45 305 ? 37 796 ? 48 406 ? 20 hsa-miR-425-3p 32 ? 3 30 ? 1 31 ? 8 25 ? 1 34 ? 13 19 ? 8 hsa-miR-425-5p 732 ? 105 597 ? 242 547 ? 152 650 ? 144 1,029 ? 156 630 ? 434 hsa-miR-454-3p 594 ? 141 518 ? 92 631 ? 28 467 ? 63 537 ? 107 311 ? 58 hsa-miR-455-3p 153 ? 21 123 ? 16 120 ? 27 123 ? 40 140 ? 35 75 ? 30 hsa-miR-466 1,389 ? 552 1,257 ? 551 907 ? 492 1,076 ? 311 1,170 ? 95 1,758 ? 407 hsa-miR-483-3p 3 ? 1 6 ? 5 27 ? 17 17 ? 13 5 ? 2 2 ? 1 hsa-miR-483-5p 204 ? 24 192 ? 14 167 ? 44 94 ? 40 90 ? 24 195 ? 41 hsa-miR-484 84 ? 39 53 ? 15 46 ? 8 38 ? 8 62 ? 18 19 ? 12 hsa-miR-486-3p 26 ? 9 16 ? 6 2 ? 2 14 ? 5 50 ? 10 14 ? 4 hsa-miR-486-5p 36 ? 7 28 ? 5 21 ? 3 16 ? 3 34 ? 12 12 ? 6 hsa-miR-489 15 ? 6 22 ? 4 16 ? 1 11 ? 6 36 ? 5 13 ? 8 hsa-miR-494 456 ? 124 451 ? 77 351 ? 32 239 ? 75 268 ? 111 568 ? 188 hsa-miR-503-5p 48 ? 7 45 ? 23 41 ? 12 18 ? 5 87 ? 9 29 ? 21 hsa-miR-505-3p 23 ? 9 33 ? 4 27 ? 4 16 ? 5 32 ? 9 16 ? 2 hsa-miR-513a-5p 9 ? 2 5 ? 2 11 ? 5 9 ? 3 18 ? 5 173 ? 83 hsa-miR-532-3p 5 ? 2 4 ? 2 3 ? 1 13 ? 20 2 ? 3 3 ? 1 hsa-miR-568 86 ? 34 111 ? 5 63 ? 15 52 ? 5 52 ? 21 57 ? 35 hsa-miR-574-3p 1,128 ? 409 1,446 ? 402 382 ? 264 563 ? 173 1,020 ? 121 804 ? 252 hsa-miR-574-5p 228 ? 48 208 ? 37 124 ? 36 169 ? 53 248 ? 16 141 ? 52 hsa-miR-576-3p 1 ? 0 4 ? 2 3 ? 3 211 ? 72 2 ? 2 30 ? 4 hsa-miR-584-5p 68 ? 17 80 ? 3 110 ? 23 57 ? 11 105 ? 19 55 ? 25  114 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-601 25 ? 4 23 ? 2 42 ? 26 7 ? 3 11 ? 9 7 ? 4 hsa-miR-603 15 ? 12 34 ? 22 12 ? 4 6 ? 2 19 ? 6 24 ? 9 hsa-miR-612 5 ? 2 5 ? 4 4 ? 3 5 ? 3 6 ? 0 38 ? 13 hsa-miR-625-3p 42 ? 7 37 ? 4 33 ? 4 20 ? 5 43 ? 10 19 ? 8 hsa-miR-625-5p 69 ? 6 73 ? 4 70 ? 6 51 ? 16 109 ? 23 55 ? 37 hsa-miR-629-5p 23 ? 12 25 ? 8 33 ? 3 22 ? 4 33 ? 3 24 ? 7 hsa-miR-638 199 ? 30 276 ? 27 197 ? 34 282 ? 40 190 ? 10 582 ? 47 hsa-miR-641 5 ? 1 14 ? 16 5 ? 3 8 ? 4 11 ? 2 8 ? 5 hsa-miR-663a 29 ? 23 44 ? 7 30 ? 8 44 ? 12 45 ? 13 117 ? 44 hsa-miR-664b-3p 23 ? 2 17 ? 8 15 ? 3 15 ? 2 24 ? 4 18 ? 16 hsa-miR-668 15 ? 21 40 ? 8 11 ? 3 17 ? 1 52 ? 32 58 ? 54 hsa-miR-671-5p 61 ? 14 55 ? 12 86 ? 21 46 ? 6 45 ? 7 40 ? 21 hsa-miR-744-5p 75 ? 22 67 ? 16 75 ? 18 51 ? 18 107 ? 30 37 ? 30 hsa-miR-762 153 ? 43 135 ? 20 98 ? 26 211 ? 46 151 ? 23 359 ? 45 hsa-miR-765 154 ? 56 238 ? 33 200 ? 44 203 ? 63 99 ? 15 158 ? 28 hsa-miR-877-5p 86 ? 29 58 ? 10 58 ? 13 59 ? 25 64 ? 6 30 ? 20 hsa-miR-885-5p 2 ? 2 3 ? 2 23 ? 13 9 ? 9 3 ? 2 2 ? 1 hsa-miR-936 13 ? 4 19 ? 7 8 ? 2 17 ? 5 6 ? 5 33 ? 21 hsa-miR-937-3p 6 ? 4 4 ? 3 30 ? 19 16 ? 6 3 ? 1 4 ? 1 hsa-miR-937-5p 40 ? 13 49 ? 16 103 ? 51 136 ? 60 24 ? 5 102 ? 78 hsa-miR-1180 14 ? 2 16 ? 7 38 ? 11 45 ? 7 18 ? 3 9 ? 5 hsa-miR-1183 5 ? 5 6 ? 3 32 ? 9 34 ? 11 5 ? 4 4 ? 0 hsa-miR-1227-5p 27 ? 5 32 ? 3 29 ? 10 51 ? 10 17 ? 5 57 ? 27 hsa-miR-1228-3p 5 ? 4 5 ? 7 15 ? 13 21 ? 17 7 ? 4 4 ? 1 hsa-miR-1228-5p 50 ? 6 46 ? 5 44 ? 7 41 ? 2 19 ? 1 19 ? 7 hsa-miR-1229-5p 9 ? 6 10 ? 5 13 ? 7 6 ? 1 8 ? 6 20 ? 15 hsa-miR-1233-1-5p 26 ? 13 41 ? 14 30 ? 9 48 ? 15 24 ? 2 79 ? 23  115 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-1234-5p 392 ? 39 444 ? 40 413 ? 40 384 ? 35 276 ? 18 770 ? 208 hsa-miR-1236-3p 0 ? 1 3 ? 3 32 ? 18 8 ? 8 1 ? 1 1 ? 0 hsa-miR-1237-5p 39 ? 33 68 ? 6 44 ? 16 59 ? 12 45 ? 17 219 ? 41 hsa-miR-1246 897 ? 104 824 ? 107 876 ? 77 1,385 ? 680 929 ? 71 5,073 ? 1,939 hsa-miR-1260a 10 ? 10 17 ? 10 22 ? 4 18 ? 14 12 ? 7 30 ? 14 hsa-miR-1260b 386 ? 224 423 ? 51 433 ? 127 728 ? 68 744 ? 231 1,645 ? 371 hsa-miR-1268a 27 ? 9 25 ? 9 90 ? 24 107 ? 33 11 ? 6 16 ? 2 hsa-miR-1268b 46 ? 9 56 ? 16 140 ? 43 147 ? 64 70 ? 12 71 ? 26 hsa-miR-1273c 6 ? 6 11 ? 2 4 ? 3 6 ? 5 4 ? 1 26 ? 13 hsa-miR-1273f 16 ? 3 14 ? 0 9 ? 3 13 ? 1 14 ? 4 60 ? 12 hsa-miR-1273g-3p 920 ? 234 870 ? 556 797 ? 313 2,510 ? 346 1,153 ? 253 4,219 ? 1,209 hsa-miR-1275 214 ? 35 216 ? 20 207 ? 5 142 ? 7 260 ? 8 817 ? 314 hsa-miR-1281 5 ? 3 7 ? 3 28 ? 8 29 ? 15 6 ? 7 4 ? 1 hsa-miR-1305 15 ? 20 4 ? 3 4 ? 3 12 ? 6 3 ? 4 5 ? 2 hsa-miR-1306-3p 9 ? 4 5 ? 1 24 ? 9 25 ? 5 3 ? 1 3 ? 1 hsa-miR-1307-3p 96 ? 31 79 ? 16 116 ? 15 141 ? 8 117 ? 32 66 ? 13 hsa-miR-1469 124 ? 27 81 ? 17 107 ? 29 137 ? 40 89 ? 17 152 ? 27 hsa-miR-1538 6 ? 5 8 ? 4 4 ? 1 15 ? 21 4 ? 1 12 ? 4 hsa-miR-1587 25 ? 8 25 ? 9 82 ? 37 121 ? 76 25 ? 3 120 ? 30 hsa-miR-1825 4 ? 4 6 ? 6 20 ? 5 15 ? 18 6 ? 4 1 ? 1 hsa-miR-1908 7 ? 4 7 ? 4 3 ? 2 17 ? 20 7 ? 4 4 ? 2 hsa-miR-1915-3p 24 ? 8 35 ? 2 16 ? 1 47 ? 13 28 ? 3 122 ? 23 hsa-miR-1972 6 ? 1 49 ? 15 6 ? 2 28 ? 3 8 ? 6 7 ? 1 hsa-miR-1973 208 ? 38 386 ? 23 226 ? 68 507 ? 52 293 ? 23 799 ? 229 hsa-miR-2392 18 ? 5 12 ? 2 17 ? 4 26 ? 4 7 ? 6 87 ? 69 hsa-miR-2861 22 ? 14 19 ? 5 22 ? 6 22 ? 4 9 ? 1 37 ? 13 hsa-miR-3064-3p 14 ? 13 23 ? 8 19 ? 6 15 ? 6 24 ? 8 18 ? 4  116 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-3065-5p 12 ? 3 15 ? 5 11 ? 2 21 ? 2 12 ? 6 22 ? 10 hsa-miR-3127-5p 6 ? 5 4 ? 2 1 ? 1 490 ? 142 4 ? 3 63 ? 17 hsa-miR-3141 280 ? 43 245 ? 25 260 ? 89 166 ? 59 109 ? 25 114 ? 42 hsa-miR-3145-3p 2 ? 2 3 ? 4 1 ? 1 114 ? 33 3 ? 3 5 ? 3 hsa-miR-3146 2 ? 2 4 ? 4 1 ? 1 322 ? 116 2 ? 2 18 ? 7 hsa-miR-3149 65 ? 15 68 ? 4 78 ? 16 59 ? 6 54 ? 13 48 ? 15 hsa-miR-3150b-3p 70 ? 73 74 ? 73 27 ? 17 46 ? 11 66 ? 56 91 ? 50 hsa-miR-3162-5p 3 ? 3 2 ? 3 1 ? 1 2 ? 1 2 ? 0 27 ? 16 hsa-miR-3164 0 ? 0 2 ? 1 1 ? 1 183 ? 43 4 ? 4 11 ? 5 hsa-miR-3178 693 ? 73 646 ? 58 726 ? 153 892 ? 181 1,256 ? 352 2,549 ? 1,059 hsa-miR-3182 44 ? 10 38 ? 1 45 ? 8 44 ? 25 33 ? 4 123 ? 59 hsa-miR-3185 9 ? 2 8 ? 2 4 ? 2 8 ? 0 14 ? 3 32 ? 8 hsa-miR-3195 19 ? 3 14 ? 3 12 ? 4 15 ? 6 26 ? 11 28 ? 19 hsa-miR-3196 260 ? 43 293 ? 32 167 ? 21 278 ? 36 219 ? 49 740 ? 137 hsa-miR-3197 29 ? 12 55 ? 4 14 ? 3 145 ? 64 62 ? 33 132 ? 90 hsa-miR-3591-3p 123 ? 44 156 ? 21 115 ? 7 103 ? 19 69 ? 33 146 ? 60 hsa-miR-3607-3p 31 ? 3 41 ? 8 22 ? 2 17 ? 7 59 ? 9 31 ? 14 hsa-miR-3607-5p 161 ? 7 179 ? 30 99 ? 28 143 ? 49 331 ? 88 215 ? 266 hsa-miR-3609 95 ? 19 93 ? 13 82 ? 10 63 ? 14 94 ? 19 85 ? 52 hsa-miR-3610 33 ? 5 33 ? 3 27 ? 13 134 ? 46 10 ? 2 28 ? 12 hsa-miR-3612 1 ? 1 4 ? 3 1 ? 2 235 ? 84 0 ? 0 16 ? 7 hsa-miR-3613-3p 19 ? 4 10 ? 1 14 ? 7 21 ? 17 14 ? 9 18 ? 9 hsa-miR-3620-5p 40 ? 17 44 ? 12 98 ? 21 142 ? 42 31 ? 9 149 ? 31 hsa-miR-3621 11 ? 11 7 ? 3 4 ? 0 10 ? 10 1 ? 1 25 ? 17 hsa-miR-3622b-5p 4 ? 1 3 ? 2 2 ? 2 3 ? 3 3 ? 2 20 ? 12 hsa-miR-3651 11 ? 8 16 ? 4 9 ? 0 22 ? 4 18 ? 9 42 ? 15 hsa-miR-3652 4 ? 4 8 ? 7 33 ? 9 38 ? 16 2 ? 1 7 ? 2  117 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-3656 159 ? 2 135 ? 26 254 ? 59 308 ? 107 113 ? 27 365 ? 40 hsa-miR-3661 4 ? 2 4 ? 3 2 ? 2 3 ? 4 7 ? 4 19 ? 14 hsa-miR-3663-3p 29 ? 12 36 ? 7 30 ? 10 23 ? 12 21 ? 4 23 ? 7 hsa-miR-3665 739 ? 283 948 ? 86 799 ? 54 1,442 ? 80 908 ? 151 2,545 ? 350 hsa-miR-3667-5p 19 ? 3 20 ? 1 23 ? 15 6 ? 5 3 ? 2 15 ? 6 hsa-miR-3673 1 ? 1 2 ? 1 0 ? 0 153 ? 34 2 ? 1 18 ? 5 hsa-miR-3676-3p 147 ? 105 234 ? 66 89 ? 12 109 ? 30 90 ? 23 12 ? 3 hsa-miR-3676-5p 97 ? 4 88 ? 37 98 ? 20 211 ? 102 86 ? 5 291 ? 61 hsa-miR-3935 6 ? 4 15 ? 7 13 ? 1 31 ? 3 4 ? 1 25 ? 7 hsa-miR-3940-5p 100 ? 12 132 ? 3 150 ? 40 164 ? 70 105 ? 32 252 ? 102 hsa-miR-3960 1,187 ? 199 1,365 ? 44 1,072 ? 69 1,580 ? 55 966 ? 305 3,014 ? 260 hsa-miR-4254 9 ? 2 9 ? 6 30 ? 16 24 ? 13 10 ? 1 8 ? 2 hsa-miR-4267 1,540 ? 1,341 1,758 ? 1,759 621 ? 237 1,120 ? 515 4,242 ? 3,592 5,950 ? 1,298 hsa-miR-4270 22 ? 5 19 ? 3 15 ? 6 29 ? 1 11 ? 4 66 ? 10 hsa-miR-4280 16 ? 5 31 ? 21 9 ? 9 29 ? 22 7 ? 5 13 ? 8 hsa-miR-4281 214 ? 84 198 ? 24 224 ? 38 136 ? 1 106 ? 12 147 ? 56 hsa-miR-4284 52 ? 11 91 ? 25 46 ? 11 101 ? 13 55 ? 18 256 ? 24 hsa-miR-4286 47 ? 8 49 ? 22 75 ? 37 57 ? 14 50 ? 14 67 ? 25 hsa-miR-4288 624 ? 301 605 ? 203 924 ? 387 428 ? 371 353 ? 158 1,000 ? 771 hsa-miR-4289 104 ? 65 159 ? 22 105 ? 23 49 ? 28 88 ? 26 98 ? 54 hsa-miR-4298 252 ? 41 251 ? 13 295 ? 78 140 ? 38 141 ? 32 113 ? 14 hsa-miR-4301 84 ? 32 96 ? 49 117 ? 68 180 ? 100 210 ? 148 613 ? 289 hsa-miR-4306 86 ? 13 76 ? 3 89 ? 10 53 ? 9 79 ? 4 39 ? 11 hsa-miR-4314 1 ? 1 2 ? 2 0 ? 0 139 ? 31 0 ? 1 13 ? 7 hsa-miR-4317 29 ? 7 23 ? 23 30 ? 20 13 ? 15 13 ? 14 71 ? 60 hsa-miR-4321 20 ? 18 25 ? 11 6 ? 4 14 ? 10 10 ? 5 21 ? 15 hsa-miR-4324 2,900 ? 555 2,742 ? 223 3,156 ? 449 2,397 ? 742 2,170 ? 291 2,239 ? 459  118 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-4325 28 ? 7 29 ? 15 38 ? 28 25 ? 30 18 ? 8 75 ? 58 hsa-miR-4329 12 ? 19 1 ? 1 1 ? 2 4 ? 2 1 ? 1 4 ? 3 hsa-miR-4419b 228 ? 31 233 ? 36 220 ? 94 191 ? 127 91 ? 32 75 ? 30 hsa-miR-4421 2 ? 0 2 ? 1 0 ? 1 99 ? 29 1 ? 2 11 ? 2 hsa-miR-4429 368 ? 33 340 ? 61 459 ? 113 441 ? 98 221 ? 76 217 ? 105 hsa-miR-4433-3p 17 ? 4 25 ? 2 30 ? 3 40 ? 10 18 ? 5 80 ? 33 hsa-miR-4440 3 ? 0 2 ? 2 17 ? 7 19 ? 16 2 ? 2 2 ? 0 hsa-miR-4442 72 ? 28 82 ? 8 71 ? 34 32 ? 10 33 ? 6 39 ? 13 hsa-miR-4443 2,051 ? 586 2,466 ? 42 2,259 ? 204 4,197 ? 276 2,223 ? 229 2,815 ? 216 hsa-miR-4444 46 ? 9 44 ? 12 55 ? 12 9 ? 4 26 ? 5 8 ? 2 hsa-miR-4447 67 ? 30 89 ? 10 90 ? 33 44 ? 29 62 ? 20 177 ? 153 hsa-miR-4454 211 ? 94 238 ? 23 215 ? 68 393 ? 103 382 ? 33 1,158 ? 478 hsa-miR-4455 251 ? 44 215 ? 19 321 ? 106 136 ? 30 177 ? 12 99 ? 13 hsa-miR-4459 353 ? 47 423 ? 30 366 ? 79 253 ? 66 225 ? 62 277 ? 49 hsa-miR-4462 6 ? 4 6 ? 2 33 ? 14 26 ? 12 2 ? 2 6 ? 2 hsa-miR-4463 40 ? 10 51 ? 2 71 ? 11 119 ? 17 37 ? 6 267 ? 36 hsa-miR-4466 101 ? 21 97 ? 14 67 ? 11 109 ? 8 77 ? 10 190 ? 34 hsa-miR-4472 64 ? 14 87 ? 5 70 ? 40 38 ? 15 41 ? 6 224 ? 154 hsa-miR-4481 12 ? 6 16 ? 10 118 ? 37 114 ? 49 5 ? 1 5 ? 1 hsa-miR-4482-5p 2 ? 2 1 ? 1 0 ? 0 128 ? 48 2 ? 1 9 ? 6 hsa-miR-4484 247 ? 57 225 ? 33 172 ? 20 429 ? 81 149 ? 20 1,680 ? 111 hsa-miR-4485 44 ? 20 96 ? 64 35 ? 11 1,217 ? 362 158 ? 27 1,703 ? 1,335 hsa-miR-4486 19 ? 6 34 ? 2 22 ? 3 42 ? 9 17 ? 8 44 ? 25 hsa-miR-4488 274 ? 60 308 ? 13 307 ? 26 336 ? 33 354 ? 79 644 ? 81 hsa-miR-4492 21 ? 6 19 ? 7 14 ? 3 36 ? 6 31 ? 4 108 ? 6 hsa-miR-4497 393 ? 107 466 ? 12 484 ? 92 531 ? 58 629 ? 110 1,333 ? 250 hsa-miR-4499 69 ? 11 50 ? 15 57 ? 9 33 ? 3 28 ? 4 31 ? 12  119 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-4500 14 ? 9 10 ? 8 31 ? 31 12 ? 18 7 ? 6 67 ? 65 hsa-miR-4505 27 ? 6 35 ? 2 26 ? 6 63 ? 9 46 ? 9 259 ? 30 hsa-miR-4507 22 ? 4 26 ? 8 64 ? 26 91 ? 32 23 ? 9 113 ? 17 hsa-miR-4508 387 ? 51 341 ? 52 427 ? 19 577 ? 102 595 ? 131 1,565 ? 615 hsa-miR-4514 22 ? 8 19 ? 9 10 ? 7 10 ? 2 44 ? 16 177 ? 107 hsa-miR-4516 472 ? 157 585 ? 112 415 ? 33 645 ? 27 442 ? 109 1,439 ? 262 hsa-miR-4521 886 ? 204 1,055 ? 185 785 ? 67 504 ? 83 645 ? 144 61 ? 19 hsa-miR-4530 117 ? 26 128 ? 12 91 ? 17 126 ? 27 93 ? 29 499 ? 186 hsa-miR-4532 32 ? 11 31 ? 4 17 ? 8 22 ? 9 32 ? 5 165 ? 104 hsa-miR-4534 81 ? 6 66 ? 15 87 ? 21 72 ? 32 50 ? 10 63 ? 15 hsa-miR-4632-5p 25 ? 11 28 ? 15 121 ? 48 157 ? 35 13 ? 5 32 ? 14 hsa-miR-4638-5p 1,549 ? 875 1,737 ? 368 957 ? 676 1,198 ? 1,088 533 ? 410 2,963 ? 3,573 hsa-miR-4644 24 ? 5 29 ? 16 23 ? 6 24 ? 10 11 ? 5 49 ? 10 hsa-miR-4646-5p 50 ? 8 46 ? 6 55 ? 15 15 ? 7 24 ? 6 21 ? 2 hsa-miR-4648 1 ? 2 1 ? 1 0 ? 0 87 ? 14 5 ? 3 7 ? 3 hsa-miR-4649-5p 5 ? 3 6 ? 2 18 ? 13 41 ? 22 4 ? 3 52 ? 7 hsa-miR-4651 7 ? 1 12 ? 7 84 ? 33 86 ? 52 3 ? 2 10 ? 3 hsa-miR-4660 1 ? 2 4 ? 4 1 ? 1 317 ? 91 2 ? 1 21 ? 9 hsa-miR-4667-5p 47 ? 5 59 ? 12 47 ? 20 28 ? 19 18 ? 6 52 ? 20 hsa-miR-4668-5p 27 ? 20 4 ? 1 4 ? 1 29 ? 41 10 ? 1 31 ? 10 hsa-miR-4669 3 ? 2 3 ? 3 5 ? 3 31 ? 4 4 ? 5 5 ? 4 hsa-miR-4687-3p 155 ? 34 191 ? 24 160 ? 25 157 ? 36 118 ? 20 301 ? 172 hsa-miR-4690-5p 1,397 ? 739 2,599 ? 1,568 1,050 ? 261 2,301 ? 342 2,467 ? 1,775 2,483 ? 628 hsa-miR-4695-5p 13 ? 6 20 ? 6 16 ? 6 15 ? 7 10 ? 7 18 ? 14 hsa-miR-4707-5p 49 ? 13 66 ? 5 43 ? 6 56 ? 24 38 ? 5 100 ? 34 hsa-miR-4710 9 ? 4 12 ? 7 50 ? 12 61 ? 36 5 ? 2 13 ? 6 hsa-miR-4712-3p 11 ? 13 9 ? 11 7 ? 3 6 ? 8 9 ? 10 48 ? 57  120 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-4717-3p 7 ? 3 6 ? 3 27 ? 4 33 ? 8 4 ? 1 2 ? 0 hsa-miR-4724-5p 1 ? 1 1 ? 1 1 ? 1 12 ? 20 1 ? 1 1 ? 1 hsa-miR-4728-5p 13 ? 2 27 ? 5 20 ? 4 19 ? 1 13 ? 2 21 ? 5 hsa-miR-4732-5p 21 ? 6 32 ? 10 18 ? 5 11 ? 3 6 ? 2 10 ? 3 hsa-miR-4734 2,588 ? 3,267 1,951 ? 2,090 603 ? 251 1,991 ? 736 3,131 ? 3,228 7,247 ? 3,484 hsa-miR-4739 258 ? 34 282 ? 27 264 ? 36 125 ? 26 168 ? 55 170 ? 35 hsa-miR-4743-5p 23 ? 10 36 ? 5 19 ? 8 13 ? 4 6 ? 3 20 ? 10 hsa-miR-4763-3p 9 ? 4 13 ? 3 50 ? 25 51 ? 13 5 ? 2 13 ? 6 hsa-miR-4764-3p 26 ? 36 78 ? 19 17 ? 7 34 ? 36 29 ? 36 343 ? 296 hsa-miR-4778-5p 581 ? 154 643 ? 135 744 ? 165 634 ? 269 222 ? 47 293 ? 91 hsa-miR-4787-5p 156 ? 19 221 ? 31 191 ? 4 346 ? 31 267 ? 33 1,208 ? 91 hsa-miR-4800-3p 5 ? 4 6 ? 1 3 ? 2 9 ? 4 4 ? 3 85 ? 28 hsa-miR-4800-5p 43 ? 12 31 ? 5 45 ? 22 85 ? 19 14 ? 3 41 ? 10 hsa-miR-5001-5p 22 ? 12 38 ? 8 29 ? 3 55 ? 7 26 ? 9 129 ? 70 hsa-miR-5088 4 ? 3 2 ? 0 4 ? 2 135 ? 45 4 ? 3 13 ? 7 hsa-miR-5096 395 ? 107 1,459 ? 1,964 250 ? 63 632 ? 148 476 ? 134 1,283 ? 824 hsa-miR-5100 50 ? 8 57 ? 8 47 ? 22 82 ? 19 74 ? 6 133 ? 35 hsa-miR-5194 1 ? 2 5 ? 2 4 ? 2 403 ? 140 1 ? 1 60 ? 20 hsa-miR-5196-5p 13 ? 7 13 ? 6 10 ? 2 9 ? 1 6 ? 5 118 ? 65 hsa-miR-5572 7 ? 3 9 ? 3 16 ? 10 26 ? 22 7 ? 4 17 ? 9 hsa-miR-5581-5p 0 ? 1 4 ? 4 0 ? 0 115 ? 48 2 ? 3 14 ? 6 hsa-miR-5739 19 ? 5 22 ? 12 8 ? 3 263 ? 109 9 ? 6 58 ? 25 hsa-miR-5787 119 ? 7 131 ? 16 105 ? 7 116 ? 12 91 ? 35 307 ? 145 hsa-miR-6073 13 ? 3 26 ? 5 18 ? 8 24 ? 26 24 ? 24 241 ? 194 hsa-miR-6075 4 ? 3 6 ? 5 2 ? 2 3 ? 3 4 ? 1 24 ? 19 hsa-miR-6076 47 ? 3 53 ? 5 43 ? 8 27 ? 6 29 ? 6 24 ? 5 hsa-miR-6082 25 ? 5 36 ? 7 21 ? 3 13 ? 2 20 ? 10 23 ? 12  121 MiR 3 H CONTROL 3 H TPEN 12 H CONTROL 12 H TPEN 24 H CONTROL 24 H TPEN hsa-miR-6085 69 ? 15 75 ? 14 57 ? 4 87 ? 18 61 ? 2 266 ? 59 hsa-miR-6086 45 ? 12 62 ? 9 37 ? 13 23 ? 2 22 ? 4 24 ? 12 hsa-miR-6087 1,005 ? 42 1,239 ? 167 836 ? 86 1,024 ? 5 764 ? 142 2,070 ? 138 hsa-miR-6088 138 ? 48 122 ? 8 179 ? 31 121 ? 20 71 ? 14 181 ? 53 hsa-miR-6089 1,276 ? 92 1,416 ? 45 1,306 ? 63 1,761 ? 231 1,455 ? 212 2,847 ? 637 hsa-miR-6090 1,123 ? 432 1,418 ? 144 976 ? 275 1,408 ? 78 902 ? 342 2,232 ? 841 hsa-miR-6124 163 ? 30 144 ? 15 117 ? 31 163 ? 23 71 ? 10 184 ? 44 hsa-miR-6125 288 ? 26 263 ? 36 204 ? 28 240 ? 45 210 ? 33 617 ? 119 hsa-miR-6126 147 ? 44 163 ? 51 117 ? 30 382 ? 44 159 ? 30 801 ? 154 hsa-miR-6127 20 ? 10 11 ? 2 37 ? 17 43 ? 3 5 ? 4 32 ? 12 hsa-miR-6128 0 ? 1 1 ? 1 0 ? 1 61 ? 19 1 ? 1 10 ? 3 hsa-miR-6132 7 ? 2 7 ? 4 6 ? 3 13 ? 2 8 ? 3 60 ? 19 hsa-miR-6133 532 ? 202 352 ? 279 253 ? 96 373 ? 180 583 ? 269 720 ? 162 hsa-miR-6165 55 ? 16 64 ? 7 58 ? 10 41 ? 8 27 ? 7 51 ? 28 hsa-miR-6509-5p 2 ? 2 2 ? 1 7 ? 1 93 ? 25 2 ? 0 7 ? 5 hsa-miR-6510-5p 1,094 ? 231 1,233 ? 104 850 ? 161 985 ? 46 776 ? 195 1,439 ? 358 hsa-miR-6511a-5p 3 ? 3 5 ? 4 6 ? 1 3 ? 3 5 ? 7 20 ? 21 hsa-miR-6511b-5p 6 ? 3 4 ? 3 6 ? 2 7 ? 4 3 ? 4 20 ? 19 hsa-miR-6716-5p 2 ? 1 3 ? 2 1 ? 1 217 ? 89 4 ? 1 27 ? 4 hsa-miR-6722-3p 45 ? 9 57 ? 14 124 ? 49 141 ? 77 17 ? 2 85 ? 60 hsa-miR-6723-5p 5 ? 3 3 ? 1 6 ? 2 9 ? 1 9 ? 6 21 ? 18 hsa-miR-6724-5p 50 ? 20 35 ? 8 31 ? 11 46 ? 5 24 ? 3 69 ? 18  122   Figure A.1: Hsa-miR-16-5p expression measured by microarray (z-scores of log transformed signal intensities).  MDA-MB-231 cells were treated with DMSO (control) or TPEN (10 ?M ?in ?DMSO) ?for ?3, ?12 and 24 h (n=3). 2-way ANOVA indicated no significant differences between groups (p<0.05). 3 12 24 -2-1012Treatment duration (hours)Expression of hsa-miR-16-5p (z-score)Control TPEN  123   Figure A.2: Hsa-miR-16-5p expression during qRT-PCR experiment 1.  MDA-MB-231 cells were treated with control (DMSO) or TPEN ?(10 ??M ?in ?DMSO) for 3, 12 and 24 h. Values are mean ? SD (n = 6 except TPEN at 12 h, where n = 5 due to removal of an outlier). Expression is reported relative to the control sample at 3 h. Different letters indicate significant differences between means (p < 0.05).  3 12 24 0.00.51.01.5Treatment duration (h)Relative abundance of hsa-mir-16-5p (fold change)ab ababab abControl TPEN  124   Figure A.3: Hsa-miR-16-5p expression during qRT-PCR experiment 2.  MDA-MB-231 cells were treated with control (DMSO) or TPEN ?(10 ??M ?in ?DMSO) ?for 3, 12 and 24 h. Values are mean ? SD (n = 6 except TPEN at 3 and 12 h, where n = 5 due to removal of outliers). Expression is reported relative to the control sample at 3 h. Different letters indicate significant differences between means (p < 0.05). 3 12 24 0.00.51.01.5Treatment duration (h)Relative abundance of hsa-mir-16-5p (fold change) ab a ab babbControl TPEN  125   Figure A.4: Hsa-miR-16-5p expression during qRT-PCR experiment 3.  MDA-MB-231 cells were treated with control (DMSO) or TPEN ?(10 ??M ?in ?DMSO) ?for 3, 12 and 24 h. Values are mean ? SD (n = 6). Expression is reported relative to the control sample at 3 h. Different letters indicate significant differences between means (p < 0.05). 3 12 24 0.00.51.01.5Treatment duration (h)Relative abundance of hsa-mir-16-5p  (fold change)a aab bab ab Control TPEN  126   Figure A.5: Hsa-miR-16-5p expression during qRT-PCR experiment 4.  MDA-MB-231 cells were treated with control (DMSO) or TPEN ?(10 ??M ?in ?DMSO) ?for 3, 12 and 24 h. Values are mean ? SD (n = 6 except Control at 3 h and TPEN at 12 h, where n = 5 due to removal of outliers). Expression is reported relative to the control sample at 3 h. Different letters indicate significant differences between means (p < 0.05). 3 12 24 0.00.51.01.5Treatment duration (h)Relative abundance of hsa-mir-16-5p (fold change) a a ab ababbControl TPEN  127    Figure A.6: Baseline DNA fragmentation in MDA-MB-231 cells at 0 h.  Apoptosis was assessed by propidium-iodide flow cytometric assay. Dot plots and histograms of propidium-iodide labeled cells are shown.! 128   Figure A.7: DNA fragmentation in MDA-MB-231 cells treated with TPEN at 3 h.  MDA-MB-231 cells were treated with control (DMSO) or TPEN (10 ?M in DMSO) for 3 h and apoptosis was assessed by propidium-iodide flow cytometric assay. Dot plots and histograms of propidium-iodide labeled cells are shown.  129   Figure A.8: DNA fragmentation in MDA-MB-231 cells treated with TPEN at 6 h.  MDA-MB-231 cells were treated with control (DMSO) or TPEN (10 ?M in DMSO) for 6 h and apoptosis was assessed by propidium-iodide flow cytometric assay. Dot plots and histograms of propidium-iodide labeled cells are shown.!  130   Figure A.9: DNA fragmentation in MDA-MB-231 cells treated with TPEN at 12 h.  MDA-MB-231 cells were treated with control (DMSO) or TPEN (10 ?M in DMSO) for 12 h and apoptosis was assessed by propidium-iodide flow cytometric assay. Dot plots and histograms of propidium-iodide labeled cells are shown.   131   Figure A.10: DNA fragmentation in MDA-MB-231 cells treated with TPEN at 24 h.  MDA-MB-231 cells were treated with control (DMSO) or TPEN (10 ?M in DMSO) for 24 h and apoptosis was assessed by propidium-iodide flow cytometric assay. Dot plots and histograms of propidium-iodide labeled cells are shown.  132   Figure A.11: 48-h TPEN-induced DNA fragmentation in MDA-MB-231 cells.  MDA-MB-231 cells were treated with control (DMSO) or TPEN (10 ?M in DMSO) for 48 h and apoptosis was assessed by propidium-iodide flow cytometric assay. Dot plots and histograms of propidium-iodide labeled cells are shown.!  133    Figure A.12: 72-h TPEN-induced DNA fragmentation in MDA-MB-231 cells.  MDA-MB-231 cells were treated with control (DMSO) or TPEN (10 ?M in DMSO) for 72 h and apoptosis was assessed by propidium-iodide flow cytometric assay. Dot plots and histograms of propidium-iodide labeled cells are shown.! 

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