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BMC Cancer. 2019 Nov 21;19(1):1130
The Author(s).
Daoud, Afra Z
Mulholland, Eoghan J
Cole, Grace
McCarthy, Helen O
A severe lack of early diagnosis coupled with resistance to most available therapeutic options renders pancreatic cancer as a major clinical concern. The limited efficacy of current treatments necessitates the development of novel therapeutic strategies that are based on an understanding of the molecular mechanisms involved in pancreatic cancer progression. MicroRNAs (miRNAs) are non-coding small RNAs that regulate the expression of multiple proteins in the post-translation process and thus have promise as biomarkers, prognostic agents, and as advanced pancreatic therapies. Profiling of deregulated miRNAs in pancreatic cancer can correlate to diagnosis, indicate optimal treatment and predict response to therapy. Furthermore, understanding the main effector genes in pancreatic cancer along with downstream pathways can identify possible miRNAs as therapeutic candidates. Additionally, obstacles to the translation of miRNAs into the clinic are also considered. Distinct miRNA expression profiles can correlate to stages of malignant pancreatic disease, and hold potential as biomarkers, prognostic markers and clinical targets. However, a limited understanding and validation of the specific role of such miRNAs stunts clinical application. Target prediction using algorithms provides a wide range of possible targets, but these miRNAs still require validation through pre-clinical studies to determine the knock-on genetic effects.
DEBATE Open AccessMicroRNAs in Pancreatic Cancer:biomarkers, prognostic, and therapeuticmodulatorsAfra Z. Daoud1†, Eoghan J. Mulholland2†, Grace Cole3,4 and Helen O. McCarthy1*AbstractA severe lack of early diagnosis coupled with resistance to most available therapeutic options renders pancreaticcancer as a major clinical concern. The limited efficacy of current treatments necessitates the development of noveltherapeutic strategies that are based on an understanding of the molecular mechanisms involved in pancreaticcancer progression. MicroRNAs (miRNAs) are non-coding small RNAs that regulate the expression of multipleproteins in the post-translation process and thus have promise as biomarkers, prognostic agents, and as advancedpancreatic therapies.Profiling of deregulated miRNAs in pancreatic cancer can correlate to diagnosis, indicate optimal treatment andpredict response to therapy. Furthermore, understanding the main effector genes in pancreatic cancer along withdownstream pathways can identify possible miRNAs as therapeutic candidates. Additionally, obstacles to thetranslation of miRNAs into the clinic are also considered.Distinct miRNA expression profiles can correlate to stages of malignant pancreatic disease, and hold potential asbiomarkers, prognostic markers and clinical targets. However, a limited understanding and validation of the specificrole of such miRNAs stunts clinical application. Target prediction using algorithms provides a wide range of possibletargets, but these miRNAs still require validation through pre-clinical studies to determine the knock-on geneticeffects.Keywords: Pancreatic Cancer, microRNA, Biomarker, Prognosis, Chemoresistance, Gene therapyBackgroundIntroduction to Pancreatic CancerCancer is a devastating disease, and of the 18 million di-agnosed globally in 2018, approximately 500,000 caseswere pancreatic [1]. Furthermore, pancreatic cancer(PC), in all forms, has the lowest survival rate of anycancer type; with less than 1% overall 10-year survival,and 3% overall 5-year survival based on statistics of pa-tients from England and Wales [2]. Although significantprogress has been made in the development of novelcancer therapies, PC survival rates have failed to im-prove in the last 40 years [2]. A fundamental reason forthis is that PC typically presents as an advanced diseasewith extensive metastatic deposits that arise in the liver[3]. Additionally, surgeons are often reluctant to conductresections on local tumours due to growth around vitalarteries and para-aortal metastases [4, 5].The evolution of genetic mutations required for me-tastasis can take over a decade to come into fruitionfrom the initial primary non-metastatic cell within thepancreas [3]. This means that if the disease is detectedwithin the first couple of years of tumorigenesis, there isa significantly improved chance of disease control witheffective treatment [6]. Abdominal pain and abnormaldigestive patterns have been reported to be recurring in70% patients with PC diagnosis, which in later stages be-come more apparent with specific patterns identified [7].Diabetes mellitus (DM) is unsurprisingly found in twothirds of PC patients. Studies consistently show an in-crease in the risk of PC development with type 2 DM[8]. Furthermore, in a recent study by Wang et al. [9],© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.* Correspondence: h.mccarthy@qub.ac.uk†Afra Z. Daoud and Eoghan J. Mulholland contributed equally to this work.1School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, NorthernIreland BT9 7BL, UKFull list of author information is available at the end of the articleDaoud et al. BMC Cancer (2019) 19:1130 https://doi.org/10.1186/s12885-019-6284-yDM was linked with increased metastasis in PC patients.These findings were linked to an increased inflammatoryresponse accompanied by high glucose levels promotingPC progression [9]. Nonetheless, the pancreatic tumouritself can be diabetogenic resulting in dysfunctional β-cells, required for insulin production [10]. This explainsthe association of PC with 10% of new-onset diabetes[11], as well as highlighting newly developed DM as apossible PC symptom.Taken together, these facts indicate that PC is still achallenge in terms of diagnosis, treatment and prognos-tic outlook. This review therefore will explore the mo-lecular changes which can occur in PC development andprogression, and how (microRNA) miRNA can play arole in terms of, disease screening, prognoses and newtherapeutic options.Main textMolecular changes during Pancreatic carcinogenesisThe development of PC is a multistage process thatinvolves alterations in well-characterised genes [12].One of the most commonly mutated oncogenes isthe Kirsten Rat Sarcoma (K-RAS) gene, detected in90% of PC cases [11].Typical PC tumour suppressor mutations can befound in cyclin-dependent kinase (CDKN2A), tumourprotein 53 (TP53), mothers against decapentaplegichomolog 3 (SMAD3), and breast cancer A2 (BRCA2)[13]. These genetic mutations result in histological andmorphological abnormalities within the ductal cells ofthe pancreas, forming papillary-like structures. Thesepapillary lesions then transform into more complicatedpreneoplastic lesions known as pancreatic intraepithe-lial neoplasia (PanIN) [14]. The formation and develop-ment of PanINs occur in a stepwise linear manner thatis concurrent with the aforementioned genetic muta-tions. As a result, more complicated structures develop,progressing to the invasive (carcinoma) tumour cells(Fig. 1). Thus, PanINs are classified based upon cellularmorphology, as PanIN-1A, −1B, − 2 or − 3. PanIN-1Aand PanIN-1B are more elongated compared to thenormal ductal cells and cells with flat structures fallunder the PanIN-1A subgroup. Both A and B sub-groups are linked by the early mutation of the K-RASoncogene. With the inactivation mutation of CDKN2A,the lesions progress and acquire moderate nuclear ab-normality, forming PanIN-2. Ultimately, nuclear atypiaensues with the budding of cells into the ductal-likestructure in the late lesions known as PanIN-3. Theselate lesions arise as a result of inhibitory mutations ofp-16 and SMAD-4 and are also dubbed as in situ car-cinoma [15]. PanINs can also be divided into low-gradeor high-grade, with a high rate of occurrence of low-Fig. 1 Schematic representation of the development of PC and associated genes in each stage. The precursor lesions (PanINs) give rise to PC in amultistage process that is mediated through consecutive genetic mutations starting with early K-RAS oncogenic activation and ending inmultiple tumour suppressors silencing. Source: MS PowerpointDaoud et al. BMC Cancer (2019) 19:1130 Page 2 of 13grade in the early stages of PC and excessive tumourinvasion happening in the later stages [16].Oncogene activationK-RAS is a member of the Ras family, a guanosine tri-phosphate (GTP) family of bound proteins. Active Rasproteins bind to GTP and are responsible for prolifera-tion, cell survival and cytoskeletal remodelling throughthe regulation of several downstream modulators. Theactivity of Ras is then terminated through the hydrolysisof GTP into guanosine diphosphate (GDP), mediated byGTPase activating protein (GAP) [17].Mutations arise in the K-RAS oncogene in glycine 12(G12), glycine (G13), or glutamine (Q61L) resulting insustained K-RAS activity through maintaining the GTP-bound active form of the protein [18]. This results inpersistent activation of the downstream signalling path-ways, which will be translated in a typical cancerous cellphenotype including aberrant proliferation, microenvir-onment alterations, apoptosis suppression and cellsurvival.To study the impact of mutated K-RAS on tumourprogression, Collins et al. [19] engineered two mousemodels to have reversibly inducible K-RAS mutationthat is tissue-specific. The two models are iK-Ras* andiK-Ras-p53+/−, in which the latter has an extra inactiva-tion mutation of one allele of the p53 gene. Thesemouse models are believed to develop pancreatic lesionsin a manner mimicking human disease, with PanINs de-veloped within three weeks of K-RAS mutation induc-tion in the iK-Ras* model. This model was used todemonstrate the key role of K-RAS in the initiation ofthe tumours. On the other hand, inactivation of K-RASmutation in the iK-Ras-p53+/− model resulted in tumourregression. The same response was noted by other au-thors using the iK-Ras mouse model [20]. They have re-corded rapid tumour and stromal deterioration uponturning the K-RAS mutation off, with 50% reduction intumour mass after 48–72 h of K-RAS genetic inhibition[20]. These findings elucidate the importance of K-RASin maintaining carcinogenesis irrespective of any add-itional mutations, in a phenomenon known as “K-RASaddiction” [21]. Oncogene addiction materialises when acancer requires the sustained activation of a specificoncogene, even after the incorporation of additional gen-etic mutations through advanced stages [22]. Thisphenomenon doesn’t repudiate the importance of add-itional mutations on tumour suppressor genes in theprogression of tumours, as the tumours formed in theabsence of other tumour suppressor mutations werestable with infrequent invasiveness [20, 23]. This doesindicate that the K-RAS gene could be a potent thera-peutic intervention in pancreatic cancer.The two main downstream pathways of activated K-RAS include mitogen-activated protein kinase (MAPK)[24] and phosphoinositide-3-kinase (PI3K) [25], whichhave significant roles in promoting cell survival and pro-liferation. MAPK is a superfamily of protein kinases, in-cluding subfamilies that are identified and characterisedin mammalian cells: extracellular signal-regulated ki-nases (ERK1/ERK2), the c-Jun N-H2 terminal kinases/stress-activated protein kinase (JNK/SAPK), and the P38enzymes. Activation of the MAPK pathways is associatedwith poor prognosis in PC patients through the activa-tion of several downstream pathways. Of these pathways,ERK1/2 [26] and P38 [27] are critical downstream mod-ulators; however, the role of the P38 pathway is contra-dictory. As shown by Handa-Luca et al. who studied 99surgically resected PC, increased cytoplasmic levels ofP38 and ERK1/2 were linked with high recurrence aftersurgical resection of PC, and lower overall survival ratesof 7 months compared to 35 months for patients show-ing low cytoplasmic levels [28]. Interestingly, throughthe analysis of 36 rapid biopsies from patients, increasedlevels of P38 were linked with good prognosis. This wasevident as the postoperative survival was noted, with amedian survival rate of 27.9 months in patients showinghigh levels of P38 compared to 14.7 months in thosewith low P38 expression [29]. The samples from thisstudy were taken from patients presenting with all typesof PC, whereas the study conducted by Handa-Lucaet al. contained only pancreatic ductal adenocarcinomaspatient samples. This highlights the diversity in PC andhow molecules can have multiple roles.The progressive role of inflammatory stress accompan-ied with the tumour microenvironment is believed to beactivated through the P38 pathway [30]. Huang et al. re-corded increased proliferation and invasion of a PC cellline, PANC-1, upon targeting the β-adrenergic receptorswith the stress hormone, norepinephrine (NE), whichwas accompanied by elevated levels of the active phos-phorylated P38 [30]. Yet, Ding et al. demonstrated thatupon the inhibition of P38 MAPK pathway, ERK1/2phosphorylation increased, which translated to the en-hancement of PANC-1 proliferation [31].These findings indicate that the P38-MAPK pathwayhas a controversial role in the development of pancreaticcancer. This contradiction may be explained by theavailability of four different P38 paralogues: P38α, P38β,P38γ and P38δ (reviewed in [32]) which should be fur-ther interrogated.Tumour suppressor gene mutationsThe CDKN2A gene encoding p16 is a tumour suppres-sor commonly inactivated in pancreatic cancer (90% ofthe cases [33]). The CDKN2A gene encodes two familiesof tumour suppressor proteins. Out of these families,Daoud et al. BMC Cancer (2019) 19:1130 Page 3 of 13inhibitors of the CDK4 (INK4) family is denoted mainlyby p16 and p19 which are located on the same locus.However, these proteins are not considered to be iso-forms of each other since they are being translated fromtwo separate messenger RNAs (mRNA) produced byCDKN2A [34]. The most abundant inactivation muta-tion is believed to happen uniquely on p16. Inactivationof p16 can occur via homozygous deletion, intragenicmutations or epigenetic silencing [33, 34], which resultsin cell cycle disruption through the G1/S checkpoint in-hibition. This disruption will result in uncontrolled tran-scriptional activation which will contribute to a positivefeedback loop, leading to increased cell division and pro-liferation [35].Another mutated tumour suppressor gene is TP53 en-coding for p53, a transcription factor that is activated bycellular stress [36]. DNA damage, radiation, aberrantgrowth signals and some chemotherapeutic agents willcause cellular stress, thus activating the p53 pathway[37]. P53 activation results in cell division, inhibition, orin the case of extremely damaged cells, death throughapoptosis [36]. The p53 mutation was recorded in 70%of PC, which mainly presents as a loss of function [38].However, these mutations can also provide oncogenicfunction, as p53 silencing has been shown to increasethe expression of platelet-derived growth factor receptorb (PDGFRb) which correlates directly with invasivenessand metastasis [39, 40].Both tumour suppressor proteins p16 and p53 bindand inactivate CDK2 and 4 which impacts the cell cycleby preventing transition from G1 to S phase [41, 42].Any inactivation of these proteins will result in cell cycleprogression despite the G1/S checkpoint status ensur-ing continuous proliferation [43].Another tumour suppressor gene that is commonlyderegulated in PC is the pancreatic carcinoma 4 gene(DPC4, also known as SMAD4), which is upregulated in55% of PC [41]. Loss of SMAD4 facilitates a selectivegrowth advantage, through the regulation of the trans-forming growth factor-β (TGF-β) signalling pathway[42]. Upon activation of the TGF-β pathway, an intracel-lular cascade takes place through activation of the cellsurface serine-threonine kinase receptor type II (TβRII)which will cause activation of the type I receptorthrough phosphorylation of the glycine- and serine-richsequence (GS) domain located on the N-terminal of thetype I receptor [42]. Receptor-activated SMADs, calledR-SMADs, form trimeric complexes composed of two R-SMADs and an essential co-SMAD which is theSMAD4. These tri-complexes, translocate to the nucleusand target DNA to regulate specific genes that haveanti-mitogenic and pro-apoptotic effects [41, 43]. Theimportance of SMAD4 in all TGF-β pathways as an es-sential co-factor explains the cardinal effect of SMAD4mutations on modulating most genetic responses tothe TGF-β superfamily.Ultimately, cellular anaplasia and relapses, lymphaticinvasion, postoperative recurrence, tumour size andmetastasis to lymph nodes status have all been linkedto mutations in p53, p16, and SMAD4 [44]. Thus, allof these genetic mutations offer great potential forgene therapy interventions. The utilisation of miRNAfor the targeting of these mutations presents an op-portunity to regulate multiple pathways with a singletherapeutic [45].miRNAsMicroRNAs form a subfamily of non-coding RNAswhich regulate gene expression via mRNA degradationor translatory inhibition [46]. Approximately 50% ofmiRNAs are encoded on non-protein-coding regionsand are independently transcribed. The remaining miR-NAs are encoded on introns of protein-coding tran-scripts. In this case, miRNAs are referred to asintergenic miRNA and will be transcribed with hostgenes and processed separately to produce the matureindependent miRNA [47]. The biogenesis of miRNAs issummarised in Fig. 2.Activated miRNA is incorporated within the RNA-induced silencing complex (RISC), and this multipartwill bind to the target mRNA, typically on a conservedsite called the seed region. This region is included withinthe 3′ untranslated region (3’UTR) between nucleotide2–8, and functions as a target recognition site as thebinding between the two strands has 100% complemen-tarity [48]. Conserved seed matches are linked with moreknockout potency as a single nucleotide change altersthe possible targets by more than 50% [49].The cornerstone of miRNA binding is based on 3rules: perfect or near-perfect complementary binding inthe seed region, central region mismatches or “bulges”and the reasonable complementarity throughout the fullsequence of the miRNA with the target. It is importantto mention that complementarity through the two se-quences is particularly significant in the case of subopti-mal binding in the seed region. While the full sequenceof the miRNA has possible base mismatches, these mis-matches do not affect the functionality of the complex.In fact, it gives the miRNA more extensive mRNA targetpossibilities [48]. The importance of complementarybinding in the seed region is explained by the fact that itis the cornerstone in target recognition. Furthermore,the bulges facilitate the binding and functionality of theAgo protein, an essential component of the RISC re-quired for the assembly and guidance to target mRNAs[50]. Lastly, the manner of complementary bindingthroughout the miRNA and the target mRNA sequencewill determine the inhibitory mechanism of theDaoud et al. BMC Cancer (2019) 19:1130 Page 4 of 13translation process. If the binding occurs in a perfectcomplementary fashion throughout the whole sequence,complete degradation of the target mRNA will take place[51]. On the other hand, if few base pair mismatcheswere present during the binding process, the translationblockade will ensue through destabilisation of the targetmRNA by non-splicing mechanisms. These mechanismsinclude decapping or deadenylation which represents themost abundant mechanism of silencing [48, 52].Exosomes are extracellular vesicles which are endo-somal in basis and typically range in size between 40and 100 nm and function to secrete and carry bio-logical materials between cells [53]. MiRNA can alsobe secreted from cells within exosomes, and these ‘exo-miRNAs’ have exhibited extensive evidence ofinfluencing cancer initiation and development. For ex-ample, exosomal miRNA-301a was found to trigger theM2 polarisation of macrophages when derived fromhypoxic pancreatic cancer cells which was attributed tothe activation of the PTEN/PI3Kγ pathway [54]. Inter-estingly, studies have shown that exomiRNAs have thepotential to be novel biomarkers collected from bloodserum. Studies conducted by Pang et al. showed thatpancreatic cancer cells produce and secrete miRNA-155 within exosomes, with the function of activatingfibroblasts [55]. Furthermore, exosomes are non-immunogenic and protect biological cargo thus makefor excellent gene therapy systems. However, majorhurdles of getting exosomes into the clinic include theefficient loading of miRNA into exosomes, withFig. 2 The process of miRNA biogenesis and role in post-transcriptional suppression. The biogenesis of miRNAs commences in the nucleuswhere the RNA polymerase II transcribes the genetic sequence encoding the miRNA to produce a primary miRNA hairpin (pri-miRNA) which iscapped, polyadenylated and has a stem-loop structure. Further processing by the ribonuclease DROSHA enzyme occurs in the nucleus before theresultant 70 to 100 nt long pre-miRNA hairpin is transported to the cytoplasm via the Exportin5 protein (XPO5). Once the double stranded pre-miRNA is in the cytoplasm, RNAse DICER cleaves the molecule into two single strands, with a leading functional strand, and a passenger strand-often referred to as (*)- which will be degraded. The Ago proteins bind to the leading single stranded miRNA to form the RISC. The RISC isconsidered to be the functional unit in this process which facilitates the binding of miRNA into the targeted mRNA resulting in either translationrepression or target degradation. Source: MS PowerpointDaoud et al. BMC Cancer (2019) 19:1130 Page 5 of 13influencing factors including solubility and charge. Fur-thermore, the pathways on which any therapeutic miRNAfunctions would need to be extensively defined, to avoidundesirable off-target effects [56].miRNA in Pancreatic CancermiRNAs play a major role in carcinogenesis, falling intotwo categories: tumour suppressor miRNAs, and onco-genic miRNAs (also termed oncomiRs) [57]. The classifi-cation is dependent on the role of the target mRNAs inthe tumour initiation process. Normally, oncogenes andtumour suppressor genes are regulated at an optimal ac-tivation/inhibition equilibrium. If downregulation of aspecific miRNA increases the activity of a correspondingoncogene, this is identified as a tumour suppressormiRNA. On the other hand, if upregulation of an onco-miR takes place, it will result in a continuous inhibitionof the target tumour suppressor gene. This uncontrolledinhibition will result in the loss of controlling specifictumour formation pathways. Deregulation in any of themiRNA types will contribute to the development of tu-mours [58].Patterns of miRNA expression vary markedly be-tween cancer types; thus, the use of miRNA expressionprofiles could be used as a promising non-invasive diag-nostic markers. Furthermore, miRNA profiling shouldhave superiority over using mRNA profiles as they canrepresent many more reliable targets [59]. Identification ofa small number of miRNA has been shown to be more re-liable than the data from 16,000 mRNAs with a more ro-bust hierarchical clustering [59]. In pancreatic cancer,different patterns have been found in miRNA expressionprofiles, which have contributed to the development of amiRNANome between the normal and cancerous pan-creas [60]. Determination of these miRNA expression pro-files has been made possible through different geneprofiling methods, mainly microarrays, RNA-sequencing,and RT-PCR analysis of specimens [61]. Due to the stabil-ity of miRNA in circulation, blood screening could beemployed as an approach to detect specific miRNA whichhave been linked with stage, survival rate or aggressivenessof the disease [62–64].Variations in the results observed among differentstudies are generally thought to be caused by ethni-city and regional differences [65]. Another importantfactor is the sampling procedure, as fine needle aspir-ation (FNA) results in enriched samples with a spe-cific tumour component, disregarding other cell typesduring the microdissection process. This observationwas illustrated by the molecular differences obtainedfrom bulk samples compared to fine needle aspira-tions[66]. Comparing expression patterns among dif-ferent samples could also allude to the abundance ofmiRNAs among different cell types that are naturallyoccurring in the pancreas, as well as the tumourmicroenvironment. For example, miRNA-375 was sug-gested to be linked with the islet cells [67], as theexpression was high in normal pancreatic tissuescompared to the cancerous and inflammatory tissueswith a complete absence in representative cell lines[60].miRNA samples can also be obtained via non-invasiveroutes from, peripheral blood, saliva, urine or faeces[68]. Abue et al. conducted a study to analyse the poten-tial of miRNA-483-3p and miRNA-21 as biomarkers ofpancreatic ductal adenocarcinoma from blood plasma.These plasma samples were obtained from 32 patientspresenting with pancreatic ductal adenocarcinoma, 12patients with intraductal papillary mucinous neoplasm,and 30 healthy control individuals. The levels of thesemiRNA were evaluated using qRT-PCR, compared be-tween groups and the expression of each was linked clin-ically. The plasma expression of both miRNA-483-3pand miRNA-21 was found to be significantly higher inpancreatic ductal adenocarcinoma compared to healthycontrols (p < 0.01). The plasma expression of miRNA-483-3p was significantly higher compared to intraductalpapillary mucinous neoplasm (p < 0.05), and the expres-sion of miRNA-21 was linked to advanced stage disease(p < 0.05) with metastases in the lymph nodes and liver(p < 0.01). Indeed, miRNA expression correlated with anoverall lessened survival in those patients with pancre-atic ductal adenocarcinoma (p < 0.01) [69].miRNAs as biomarkersIdentification of early biomarkers is essential in caseswhere PC surgical resection is the only curative treat-ment. Surgery is only feasible in 15–20% of patients whohave been diagnosed with early stages of PC [70, 11].However, the postoperative complications associatedwith this surgery are frequent and cases such as chronicpancreatitis or pancreatic tuberculosis are usually hardto differentiate from cancer cases [71]. To date,the serum carbohydrate antigen 19–9 (CA 19–9) hasbeen employed as a marker for assessing clinical treat-ment efficacy in pancreatic cancer [72]. Limitations asso-ciated with CA 19–9 include ineffectiveness, lowsensitivity and low specificity, yet it is still the only FDAapproved marker in PC. Other antigens such as CEA,and CA125 were completely ineffective as early markers,but some oncologists still use them as markers of ther-apy responsiveness [73]. Therefore, the PC diagnosticbiomarker need could be met by utilising identified miR-NAs as an early screening test [74]. Advantages of usingmiRNAs include stability in serum, ease of non-invasivedetection in circulation and a convenient screeningmethod [75].Daoud et al. BMC Cancer (2019) 19:1130 Page 6 of 13Lee et al. used pancreatic cancer, paired benign pan-creatic tissue, normal pancreas, and pancreatitis tissuesalong with nine cell lines to compare miRNA expressionprofiles [90]. This was achieved through employment ofreal-time PCR profiling of > 200 miRNA precursors. Thediversity of sample types used gives the study a broaderspectrum of comparison and a chance of detecting thepremalignant changes that occur through the conversionstep from benign abnormalities to malignant tumours.Among the top aberrantly expressed miRNAs (Foldchange, P-value) in cancer samples, miRNA-424 (56.3,3.62E-08), miRNA-100 (36.9, 4.40E-06), miRNA-301(34.2, 1.11E-05), miRNA-212 (22.2, 2.00E-04) andmiRNA-125b-1 (23.2, 1.00E-04) were overexpressed,whereas miRNA- 345 (− 14.5, 1.44E-15), miRNA-142-P(15.4, 3.63E-07), and miRNA-139 (− 7.91, 6.79E-11) wereall downregulated, relative to normal pancreatic samples.Additionally, miRNA-221, miRNA-376a, and miRNA-301 were found to be localised within the tumour cellsrather than other cells in the stroma [76].MiRNA-155 and miRNA-21 were also found to haveelevated expression in the precursor lesions, linkingthem with histological progression features, with a spe-cificity of miRNA-155 as a biomarker in pancreaticjuices [77]. The top five upregulated miRNA includedmiRNA-21, miRNA-196a, miRNA-27a, miRNA-146a,and miRNA-200a as listed by Hong and Park [78] fol-lowing fine needle aspiration (FNA). In comparison, themost downregulated miRNAs included miRNA-96,miRNA-217, miRNA-141, miRNA-20a, and miRNA-29c [78]. Other upregulated candidates were demon-strated via qRT-PCR by Zhang et al., and in-clude miRNA-196a, miRNA-190, miRNA-186, miRNA-221, miRNA-222, miRNA-200b, miRNA-15b, andmiRNA-95, [79]. Further evidence also showed thatmiRNA-21, miRNA-26b, miRNA-194, miRNA-200b,miRNA-200c, miRNA-320, miRNA-374 and miRNA-429 were upregulated in PC cell lines compared to nor-mal pancreas cell lines [80].Early stage K-RAS mutations, observed in PanIN le-sions, can directly affect the levels of specific miRNAsas shown by the Cordelier group [67]. Upregulation ofmiRNA-205, miRNA-200, and miRNA-21 was detectedin early adenocarcinoma lesions, through use of aKRAS(G12D) mouse model where miRNA produc-tion could be measured in pathological and nonpatho-logic ducts. The level of increased miRNA-21expression was also proportional to the degree of mor-phological changes within the lesions [81], as quantifiedusing qRT-PCR of PanIN samples using U6 as a house-keeper gene. The mechanism of action of miRNA-21 issummarised in Fig. 3. Upregulated miRNA-372,miRNA-146a, miRNA-204, miRNA-10a, and miRNA-10b were also detected in PC cell lines (CAPAN-1 andCFPAC1) compared to human normal pancreaticductal epithelial cells, with more than 10-fold changesin the latter levels [82] observed using qRT-PCR.A further study [60] showed distinct expression levelsin normal pancreatic tissues. Candidate miRNAs:miRNA-141, miRNA-148a, miRNA-200a, miRNA-200b,miRNA-200c, miRNA-216, miRNA-217, and miRNA-375 exhibited high expression levels in the normal pan-creatic tissue compared with 33 other human tissuesanalysed in the same array. Alternatively, there were lowexpression levels with miRNA-133a, miRNA-143,miRNA-145, and miRNA-150 in cancerous tissue[60].MiRNA-216 and miRNA-217 were found to have exten-sive infiltration in normal pancreatic tissues, and negliblelevels in both cancerous tissue and pancreatic cell lines.The unique expression of these two miRNAs in normalpancreatic tissue samples compared to diseased tissuesand cell lines renders a specificity for the pancreaticabundance. A global study also showed that bothmiRNA-216 and miRNA-217 were absent from 33 dif-ferent human tissues [60]. MiRNA− 93, miRNA-196a,miRNA-196b, miRNA-203, miRNA-205, miRNA-210,miRNA− 221, miRNA-222 and miRNA-224 were upreg-ulated only in cancerous tissues and cell lines. This ob-servation is linked with a potential role in the neoplasticprocess. Special interest should be directed towardsmiRNA-196a and miRNA-196b as complete absence wasobserved in the normal and pancreatitis tissues. Thisgives a potential selectivity to pancreatic cancer as de-tailed in Table 1 [60].miRNAs as prognostic factorsProfiling miRNAs among patient samples with differentdisease characteristics and stages gives an understandingof the prognostic role of miRNAs, many of which arelisted in Table 2. It was shown in a retrospective clinicalstudy comprising 200 pancreatic ductal adenocarcinomatissue samples (Department of Hepatobiliary and Pan-creas Surgery of Seoul National University Hospital) thatin a high-risk group (median survival time = 17.2months) miRNA-574-5p, miRNA-1244, miRNA-4474-5pwere upregulated. While miRNA-574-5p, miRNA-1244,miRNA-145-*, miRNA-328, miRNA-26b*, and miRNA-4321 showed association with overall survival (OS) anddisease-free survival (DFS) [83]. Taubert and coworkers[84] have investigated the role of miRNA-155, miRNA-203, miRNA-210, miRNA-222, miRNA− 216, andmiRNA-217 in overall survival. Upregulation of the firstfour of this group showed a correlation with poor prog-nosis and overall survival, while the same effect has beennoticed with the downregulation of miRNA-217. Interms of miRNA-216, no effect has been detected in ei-ther up or downregulation. Tumour-related deathincreased 5.24-fold when the overexpression of miRNA-Daoud et al. BMC Cancer (2019) 19:1130 Page 7 of 13155, miRNA-203, miRNA-210, and miRNA-222 is com-bined [84]. miRNA-21 is also another indicator of pooroutcome [85, 86]. Increased expression of miRNA-196a-2 resulted in a lower survival time (median survivaltime = 14.3 months) compared with downregulatedexpression (median survival time = 26.5 months) [86, 87].Similar effects occurred with miRNA-219 as the mediansurvival rate was 13.6 months in the overexpressing tu-mours, and 23.8 months in the tumours with downregu-lated miRNA-219 [88].Fig. 3 Schematic representation of mechanism of miR21 in PC development. miRNA-21 has previously been shown to target the expression ofPTEN and PDCD4. Through inhibition of PTEN via miRNA-21, cell survival pathways are activated. With Akt pathway functionality heightened, theinhibition of BAD increases, which is a pro-apoptotic pathway, thus leading to a reduction in apoptosis. PTEN also inhibits angiogenesis withinthe VEGF pathway, thus miRNA-21 can enhance the establishment of new vasculature. Source: MS PowerpointTable 1 miRNAs as biomarkers in pancreatic cancer (“= high mainly in early lesions)miRNA Analysis Technique Normal Pancreatic Tissues Pancreatitis PC Cell lines Reference-20a, −29c 96, − 141, Microarray High N/A Low N/A [78]-10a, −10b, − 204, − 372, Microarray Low N/A High N/A [82]-93, −133a, − 203, − 205, − 210, − 224 Microarray Low N/A High High [60]-27a Microarray Low N/A High N/A [78]-221, −222 Microarray, qRT-PCR Low N/A High High [60, 79]-216, −217 Microarray Very high N/A Very low Very low [60, 78]-200a Microarray, TaqMan low density array, qRT-PCR Low N/A High N/A [78, 81]Microarray High Low [60]-196a Microarray, qRT-PCR Absent Absent High High [60, 78, 79]-15b, −95, −186, −190, −200b qRT-PCR Low N/A High N/A [79]-146a Microarray Low N/A High N/A [78, 82]−143, −145, −150 Microarray Low High High Absent [60]−141, −148a, −200b, −200c, −375 Microarray, TaqMan low density array, qRT-PCR Low N/A N/A High [80]−139, −142, −345 qRT-PCR Low N/A Low N/a [76]−100, −125b-1, −212, −301, −424 qRT-PCR Low N/A High N/a [76]−21 Microarray, TaqMan low density array, qRT-PCR Low N/A High “ High [77, 78, 80, 81]−155 qRT-PCR Low N/A High “ N/A [77]−205 qRT-PCR Low N/A High “ N/A [81]−375 Microarray High Low Low Absent [60]Daoud et al. BMC Cancer (2019) 19:1130 Page 8 of 13Furthermore, miRNAs could play a prolific role in theprediction of chemoresistance and responsiveness to dif-ferent therapeutic approaches. For example, severalmarkers indicate the degree of responsiveness,where miRNA-320c is a prognostic factor for gemcita-bine clinical response prediction [89]. Moreover, post-surgery survival rates were higher in patients with in-creased levels of miRNA-200c (MST = 42months, 5-yearsurvival rate = 33.5%) than the lower miRNA-200c ex-pressing individuals (MST = 19months, 5-year survivalrate = 11.2%). This observation correlated with low inva-siveness of cells after the upregulation of miRNA-200cfollowing in vitro experiments [90].Irregular expression of miRNA was found in the devel-opment of a gemcitabine-resistant cell line. miRNA-200b, miRNA-200c, let-7b, let-7c, let-7d and let-7e weredownregulated in gemcitabine-resistant cells [91]. Simi-larly, miRNA-33a is also downregulated in gemcitabine-resistant cells, and upon the restoration of normal levels,gemcitabine sensitivity was restored in the MIA-PaCa2PC cell line [92]. Increased levels of miRNA-320c ingemcitabine-resistant cells suggests incorporationthrough regulation of SWI/SNF Related Matrix Associ-ated Actin Dependent Regulator Of Chromatin Subfam-ily C Member 1 (SMARCC1), as benefits from thegemcitabine regimen were only found among the pa-tients expressing SMARCC1. miRNA-205 was used incombination with gemcitabine as a chemosensitiserthrough decreased production of caveolin-1 and Ki-67[93]. Orthotopic pancreatic mice were implanted witheither lenti-hsamiRNA205 treated MIA PaCa-2R cells,or lenti-hsamiRNAScramble treated MIA PaCa-2R cells.Following intravenous delivery of GEM-conjugated poly-meric micelles thrice weekly for two weeks at a concen-tration of 40 mg/kg, it was observed that in the lenti-hsamiRNA205 treated cells there was a significant re-duction in tumour growth. These mice bore tumourswith average volumes of 77.83 ± 21 mm, compared tomice implanted with lenti-hsamiRNAScramble treatedcells, which exhibited tumours of volumes of 172.85 ±17mm.ConclusionsDue to the aggressive nature of PC and the lack of bio-markers, miRNAs represent a promising tool to help inthe development of prediction, managing, and treatingagents to improve low survival rates. The therapeuticpotential of miRNAs can be implicated after investigat-ing downstream regulatory mechanisms observed amongdifferent molecular pathways, as some can play a tumoursuppressive role and others are oncomiRNAs. The res-toration of these miRNAs levels to that of healthy tissuecould therefore be beneficial in maintaining the en-dogenous anti-tumour regulatory mechanisms.However, large-scale clinical studies need to be exploredto establish clinical relevance of the collected data. Todate, one clinical trial is ongoing studying miRNA-25 as adiagnostic tool in PC (NCT03432624) [106]. Variations inresults among studies arise from using pancreatic speci-mens, which contain heterogenous cell populations. Thesecell types include the ductal, acinar, and islet cells, alongwith other inflammatory, fibroblastic components that willaccompany the tumour development. Prediction of targetgenes of different miRNAs could be considered the majordrawback of assigning miRNA in large scale applications,as the predictive algorithms give an enormous number oftargets for a single miRNA. Attention should be given tothe miRNA’s mechanism of action, as more innovativemethods will be required to validate the predicted targets.RNAs are gaining momentum as therapy options.MRX34, for example, is a miRNA-34 mimic encapsu-lated within a lipid-based vessel known as NOV40.MRX34 was utilised in a multicentre phase I clinicaltrial (2013) for the treatment of patients with primaryliver cancer, melanoma, lymphoma, small cell lung can-cer, multiple myeloma or renal cell carcinoma. By June2016, 99 patients were recruited onto the trial withHCC, NSCLC or pancreatic cancers. Following comple-tion of the trial, 3 patients achieved prolonged con-firmed partial responses. Moreover, 14 patientspresented with stable disease (median duration- 136days) [107]. An example of an FDA approved RNAibased therapy by Alnylam Pharmaceuticals isTable 2 miRNAs as prognostic factors and the associated targets and survival status in pancreatic cancermiRNA Expression in Pancreatic Cancer compared to normal tissue Survival Status Targets Reference-10b Upregulated Poor Survival TIP30 [94, 95]−21 Upregulated Worse Survival PTEN, PDCD4, IL-6R,CDK6 [85–87]-34a Upregulated Better Survival NOTCH, BCL2, CDK6 [96–98]−155 Upregulated Poor Survival TP53INP [99, 100]-let-7 Family Downregulated Poor Survival KRAS, HRAS, TRIM71 [101, 102]−200 Family Downregulated Better Survival E-cadherin, ZEB [91, 96, 103]−216 Downregulated Poor Survival ROCK1 [104, 105]Daoud et al. BMC Cancer (2019) 19:1130 Page 9 of 13Onpattro™ for the treatment of polyneuropathy of her-editary transthyretin-mediated amyloidosis in adults.Onpattro™ contains patisaran, which comprises asiRNA conjugated with a lipid complex. The drug’smechanism of action results in binding with the TTRprotein. The reduction in TTR protein levels in theliver results in a drop in amyloid deposits. The FDA ap-proval of Onpattro™ in August 2018 was the result of asuccessful Phase III clinical trial dubbed APOLLO. Thetrial had 225 patients enrolled, 148 of which receivedOnpattro™ once every three weeks (0.3 mg/ kg bodyweight), while the remaining patients received a placebodrug. Patients receiving Onpattro™ displayed improve-ments, with 51% of patients exhibiting an improvedquality of life (measured using the Norfolk Quality ofLife Diabetic Neuropathy (QoL-DN)), as opposed toonly 10% in the placebo control [108].MiRNAs hold potential as innovative gene therapiesbut success is heavily reliant on an efficient deliveryvector [109, 110]. If indeed miRNAs are to be used inpancreatic cancer, the delivery systems must transportthe cargo to the destination site, not evoke an im-mune response and not have prohibitive productioncosts so that wide-spread adoption of the thesenanotherapies can be realised.AbbreviationsBRCA2: Breast Cancer A2; CDKN2A: Cyclin-dependent Kinase; DM: DiabetesMellitus; DNA: Deoxyribonucleic acid; eIF4E: Eukaryotic Initiation Factor 4E;ERK: Extracellular Signal-regulated Kinase; FNA: Fine Needle Aspiration;GAP: GTPase Activating Protein; GTP: Guanosine Triphosphate; K-RAS: KirstenRat Sarcoma; LDH: Lactate Dehydrogenase; MAPK: Mitogen-activated ProteinKinase; miRNA: micro Ribonucleic acid; mRNA: messenger Ribonucleic acid;NE: Norepinephrine; oncomiR: Ongogenic micro Ribonucleic acid;PanIN: Pancreatic Intraepithelial Neoplasia; PC: Pancreatic Cancer;PDGFRb: Platelet-derived Growth Factor Receptor b; PI3K: Phosphoinositide-3-kinase; RISC: RNA-induced silencing complex; RNA: Ribonucleic acid;SMAD: Mothers Against Decapentaplegic; SMARCC1: Chromatin Subfamily CMember 1; TGFβ: Transforming Growth Factor β; TβRII: Threonic KinaseReceptor II; UTR: Untranslated RegionAcknowledgementsNot Applicable.Authors’ contributionsHM was the lead PI and refined the manuscript. AD, EM and GC drafted themanuscript. AD and EM produced all the figures. All authors read andapproved the final manuscript.FundingNo funding to declare.Availability of data and materialsNot Applicable.Ethics approval and consent to participateNot Applicable.Consent for publicationNot Applicable.Competing interestsThe authors declare that they have no competing interests.Author details1School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, NorthernIreland BT9 7BL, UK. 2Gastrointestinal Stem Cell Biology Laboratory, WellcomeTrust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.3Genome Sciences Centre, British Columbia Cancer Agency, Vancouver,British Columbia V5Z 1L3, Canada. 4Department of Pathology and LaboratoryMedicine, University of British Columbia, Vancouver V6T 2B5, Canada.Received: 27 March 2019 Accepted: 24 October 2019References1. https://www.who.int/en/news-room/fact-sheets/detail/cancer.2. “Pancreatic cancer statistics | Cancer Research UK.” [Online]. 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Pancreatic Cancer
microRNA
Biomarker
Prognosis
Chemoresistance
Gene therapy
MicroRNAs in Pancreatic Cancer: biomarkers, prognostic, and therapeutic modulators
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