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Decreased miR-192 expression in peripheral blood of asthmatic individuals undergoing an allergen inhalation… Yamamoto, Masatsugu; Singh, Amrit; Ruan, Jian; Gauvreau, Gail M; O’Byrne, Paul M; R Carlsten, Christopher; FitzGerald, J M; Boulet, Louis-Philippe; Tebbutt, Scott J Nov 21, 2012

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RESEARCH ARTICLE Open AccessDecreased miR-192 expression in peripheralblood of asthmatic individuals undergoing anallergen inhalation challengeMasatsugu Yamamoto1,2,3,4†, Amrit Singh1,2,5†, Jian Ruan1,2, Gail M Gauvreau6, Paul M O'Byrne6,Christopher R Carlsten2,3,4, J Mark FitzGerald2,3,4, Louis-Philippe Boulet7 and Scott J Tebbutt1,2,3,5*AbstractBackground: MicroRNAs are small non-coding RNAs that regulate gene expression at the post-transcriptional level.While they have been implicated in various diseases, the profile changes in allergen inhalation challenge are notclarified in human. We aimed to evaluate changes in the microRNA profiles in the peripheral blood of asthmaticsubjects undergoing allergen inhalation challenge.Results: Seven mild asthmatic subjects participated in the allergen inhalation challenge. In addition, four healthycontrol subjects (HCs) were recruited. MicroRNA profiles in peripheral blood samples (pre-challenge and 2 hourspost-challenge) were measured by the NanoString nCounter assay to determine changes in miRNA levels as theseasthmatic subjects underwent an allergen inhalation challenge. One common miRNA, miR-192, was significantlyexpressed in both comparisons; HCs vs. pre-challenge and pre- vs. post-challenge, showing that miR-192 wassignificantly under-expressed in asthmatics compared to HCs and decreased in post-challenge at an FDR of 1%.Cell-specific statistical deconvolution attributed miR-192 expression in whole blood to PBMCs. MiR-192 wastechnically validated using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showingthat the level in asthmatics (pre-challenge) was significantly lower than HCs and that post-challenge wassignificantly lower than pre-challenge. The normalized relative miR-192 expression quantified using RT-qPCR specificto PBMCs was also validated. Ontology enrichment and canonical pathway analyses for target genes suggestedseveral functions and pathways involved in immune response and cell cycle.Conclusions: The miRNA profile in peripheral blood was altered after allergen inhalation challenge. Change inmiR-192 levels may be implicated in asthma mechanisms. These results suggest that allergen inhalation challengeis a suitable method to characterize peripheral miRNA profiles and potentially elucidate the mechanism of humanasthma.Keywords: Allergen inhalation challenge, Allergy, Asthma, Blood cells, Hsa-miR-192, MicroRNAs, NanoStringnCounter assay* Correspondence: Scott.Tebbutt@hli.ubc.ca†Equal contributors1UBC James Hogg Research Centre, St. Paul’s Hospital, University of BritishColumbia, Room 166, Burrard Building, 1081 Burrard Street, Vancouver, BCV6Z 1Y6, Canada2Institute for HEART+LUNG Health, Vancouver, British Columbia, CanadaFull list of author information is available at the end of the article© 2012 Yamamoto et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.Yamamoto et al. BMC Genomics 2012, 13:655http://www.biomedcentral.com/1471-2164/13/655BackgroundExposure to allergen is one of the important factors toinduce and enhance asthma pathogenesis, which is char-acterized by reversible bronchoconstriction, airwayhyperresponsiveness and airway inflammation. Allergeninhalation challenge is a useful model to investigate thepathogenesis of allergic airway diseases [1]. The asth-matic response can be evaluated by airflow limitationwhich can be measured as forced expiratory volume in 1second (FEV1) drop using a spirometer. Recently, wehave reported differential changes in the gene expressionof peripheral blood leukocytes in atopic asthmatic indivi-duals 2 hours following allergen inhalation challenge [2],indicating the application of gene expression in bloodfor assessing the asthmatic response. As blood is thepipeline of the immune system, assessing changes in ex-pression profiles gives a comprehensive view of the sta-tus of the immune system in health and disease [3].Since allergen exposure triggers immune responses, in-cluding the production of various mediators, inflamma-tory cell proliferation and infiltration in the humanbody, the immune cells and their gene expression can begood exploratory targets to understand the mechanismsof the asthmatic response to allergen inhalationchallenge.MicroRNAs (miRNAs) are a class of small noncodingRNAs with a regulatory function on gene expression tocontrol various biological processes such as cellular pro-liferation, apoptosis and differentiation. They have beenimplicated in the pathogenesis of malignancy, cardiovas-cular and other diseases [4]. Several studies have previ-ously reported the role of miRNAs in asthma using theallergen inhalation challenge model. In the lung, miRNAprofiles were significantly changed in the experimentalmouse model mimicking acute and chronic humanasthma [5,6]. In humans, while a study comparing themiRNA profile of airway biopsies revealed no significantdifference in the expression of 227 miRNAs betweenmild asthmatics compared to healthy volunteers [7], amore recent study on cultured human bronchial epithe-lial cells revealed inherent differences in the expressionof miRNAs isolated from healthy and asthmatic subjects[8]. Changes in miRNA profiles have not been clarifiedin the human asthmatic response during the allergen in-halation challenge. In this study, we focused on themiRNA profiles in human peripheral blood andhypothesized that miRNA profiles altered by allergen in-halation challenge can be detected in peripheral blood.ResultsSubjects characteristicsSeven subjects with stable, mild atopic asthma partici-pated in the allergen inhalation challenge. Four healthycontrol subjects (HCs) were recruited to serve ascontrols. The demographics of seven subjects with mildatopic asthma and four HCs are presented in Table 1.All asthmatic subjects developed an immediate drop inFEV1 of greater than 20% (Additional file 1: Figure S1).Processing of peripheral blood for miRNA Panel CodesetPeripheral blood was drawn immediately before (pre)and after allergen inhalation (post) from asthmatics. Per-ipheral blood from HCs was collected as controls.Extracted RNA was analysed using NanoString nCounterassay. One hundred and sixty-three of the 734 miRNAsprofiled using the NanoString nCounter assay wereabove background across all samples. The data set wasnormalized to the sum of the 163 miRNAs, such thateach sample had the same total miRNA code count.MiRNAs above at least 100 code counts were retainedfor downstream analysis. The dataset (72 miRNAs for 18samples) underwent log2 transformation prior to statis-tical analysis.Differentially expressed miRNA in NanoString nCounterassayTwo independent linear models were used to determinesignificant miRNAs for the two comparisons; HCs versuspre-challenge, and pre versus post-challenge. MiR-192was significant in both comparisons at a false discoveryrate (FDR) of 1% (Figure 1). MiR-192 was down-regulated in both comparisons (Figure 2A), that is, miR-192 was significantly under-expressed in asthmatics(pre-challenge) compared to HCs and decreased follow-ing allergen inhalation challenge.MiR-192 expression in peripheral blood mononuclearcells (PBMCs)Given the heterogeneous nature of whole blood, changesin miRNA expression may not be due to changes inmiRNA expression in specific cells but due to changesin the relative cell-type frequencies. Leukocyte differen-tials were significantly different among the three groups,while complete blood counts such as erythrocytes, totalleukocytes and platelets were not significantly differentamong groups (Table 2). In order to determine whethermiR-192 expression was associated with certain cell-typefrequencies, a multiple regression of miR-192 expressiononto the cell-type frequencies was performed for eachgroup (HC, pre and post) independently. Given the smallsample size of HCs (n=4), the neutrophils, eosinophilsand basophils were combined (added) to form a granulo-cyte group whereas the lymphocytes and monocyteswere combined into a peripheral blood mononuclearcells (PBMCs) group. The regression coefficientsrepresenting the mean miR-192 expression for granulo-cytes and PBMCs were extracted for each group(Additional file 1: Figure S2). In order to determineYamamoto et al. BMC Genomics 2012, 13:655 Page 2 of 9http://www.biomedcentral.com/1471-2164/13/655whether the partials for granulocytes and PBMCs weresignificantly different between groups, a test-statistic foreach cell-type between two independent groups was cal-culated (see Methods). This statistic compares whetherthe slopes for two independent groups are different forthe same cell-type: that is, for the same increase in aparticular cell frequency, is the increase in the meanmiRNA expression greater in one group that the other.This increase is independent of changes in other cellularfrequencies. MiR-192 expression at the same frequencyof granulocytes is similar between HCs and asthmatics(pre- and post-challenge), however, the mean miR-192expression was significantly (p=0.012) higher in HCsthan in asthmatics (pre-challenge) for the same fre-quency of PBMCs independent of the frequency of gran-ulocytes (Figure 2B). Additional file 1: Figure S3 showsthe empirical distributions for these comparisons for1000 permutations. This may suggest that the decreasein miR-192 seen in whole blood (Figure 2A) may be dueto a decrease in miR-192 expression in PBMCs inde-pendent of changes in the frequency of granulocytes.Although Figure 2B shows that miR-192 expression inTable 1 Demographics of subjectsSubject Age (yr) Sex (M:F) Allergen Pre PC20(mg/ml)Post PC20(mg/ml)Allergen induced shift(pre PC20/post PC20)Asthmatics1 28 F Cat Pelt 12.8 ND ND2 34 F Cat Pelt 2.7 6.1 0.443 27 M Cat Pelt 4.5 1.8 2.54 42 F Cat Hair 5.3 8.6 0.625 23 F Cat Hair 0.3 0.2 1.56 26 F Cat Hair 5.1 1.5 3.47 49 F Cat Hair 3.6 1 3.6Mean ±SE 32.7±3.3 3.3† 1.7† 2.0±0.5Healthy controls1 33 F ND2 43 F ND3 21 M ND4 43 M NDMean ±SE 35.0±4.5ND - Not determined, SE: standard error of the mean, †: geometric mean (PC20 values are measured on a log scale).log2 fold-change-log 10 p-valuelog2 fold-change-log 10 p-valueFigure 1 Volcano plot of statistical significance against fold-change of differentially expressed miRNAs between healthy controls andasthmatics (pre-challenge) and between pre and post-challenge. Dashed line represents a false discovery rate (FDR) of 1%.Yamamoto et al. BMC Genomics 2012, 13:655 Page 3 of 9http://www.biomedcentral.com/1471-2164/13/655PBMCs decreases post-challenge which is also seen inwhole blood (Figure 2A), this change was not significant.Technical validation using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR)MiR-192 was technically validated (Figure 3A) usingRT-qPCR showing that the level in asthmatics (pre-challenge) was significantly (p < 0.05) lower than HCsand that post-challenge was significantly lower than pre-challenge.The normalized relative miR-192 expression quantifiedusing RT-qPCR was also regressed onto the relative cell-type frequencies of granulocytes and PBMCs for HC andpre-challenge in separate linear models. The regressioncoefficient for PBMCs in healthy individuals (i.e. miR-192 expression in PBMCs in HC) was significantly(p=0.036) higher than the regression coefficient forPBMCs in asthmatics at pre-challenge (i.e. miR-192expression in PBMCs in asthmatics at pre-challenge)(Figure 3B).Genes up-regulated in allergen inhalation challenge andtargeted by miR-192To clarify the suggestive mechanisms of miR-192 in al-lergen inhalation challenge, genes targeted by miR-192were retrieved from the list of differentially expressedgenes between pre- and 2 hour post-allergen challenge,which Kam et al. reported in their manuscript [2].Among 80 genes which were significantly altered andpredicted as target genes for miR-192 by databases mi-Randa and TargetScan, 56 genes were up-regulated while24 genes were down-regulated post-challenge. To clarifythe biological mechanisms, we performed network mod-eling using MetaCore from GeneGo (St Joseph, MI,USA), allowing us to build a candidate network indicat-ing possible interactions between genes. The FunctionalOntology Enrichment tool, which was used for analysinglog2 miR-192 code countAMean cellular miR-192 expression p=0.012HC Pre Post HC Pre PostBFigure 2 MiR-192 expression in whole blood and in peripheral blood mononuclear cells (PBMCs). A. MiR-192 expression in whole blood.B. Cell-specific miR-192 expression in granulocytes (gray bar) and PBMCs (white bar) comparing healthy control (HC) and asthmatics pre andpost-challenge (Pre, Post).Table 2 Complete blood count and leukocyte differentials for two comparisonsHealthy controls Pre-challenge Post-challenge P-value HC vs. pre P-value pre vs. postComplete blood countErythrocytes (1012/L) 4.41 (0.17) 4.36 (0.08) 4.40 (0.08) 0.786 0.361Platelets (109/mL) 246 (18) 258 (37) 273 (36) 0.759 0.084Leukocytes (109/mL) 5.48 (0.84) 6.10 (0.35) 6.91 (0.37) 0.199 0.071Leukocyte differentialsNeutrophils (%) 56.4 (7.8) 57.9 (4.5) 65.0 (2.6) 0.782 0.012*Lymphocytes (%) 29.6 (5.5) 29.5 (4.4) 25.6 (2.6) 0.989 0.120Monocytes (%) 4.5 (0.7) 7.4 (0.7) 5.7 (0.8) 0.042* 0.013*Eosinophils (%) 2.1 (0.6) 4.7 (0.8) 3.3 (0.7) 0.077 0.002*Basophils (%) 5.6 (5.5) 0.5 (0.1) 0.4 (0.1) 0.002* 1.000HC, healthy controls. pre, pre-challenge (asthmatics). post, post-challenge (asthmatics). Values reported as mean (standard error of the mean). *: p < 0.05.Yamamoto et al. BMC Genomics 2012, 13:655 Page 4 of 9http://www.biomedcentral.com/1471-2164/13/65556 up-regulated genes by considering their mappingsonto terms of a given MetaCore ontology, showed anumber of significant (p<0.01) inflammatory pathwayssuch as ChREBP regulation pathway and IFNα/β signal-ling pathway (Additional file 1: Table S1). Similarly, thelist of up-regulated target genes was used to generatebiological networks using the Canonical Pathway Model-ing algorithm. Additional file 1: Table S2 shows the listof networks that are highly enriched with canonicalpathways. The results comprise network objects corre-sponding to genes and other interacting objects in ca-nonical pathways found in databases. For the top twohighly enriched pathways, one pathway was related toDNA damage and cell cycle regulation (Additional file1: Figure S4A) and the other pathway was related toimmunological response and stress response (Additionalfile 1: Figure S4B).DiscussionWe analysed for the first time the change in miRNAprofiles of human asthmatics undergoing allergen inhal-ation challenge as well as the difference in miRNA pro-files between pre-level in asthmatics and healthy controlsubjects without asthma history. MiR-192 was signifi-cantly lower in post-allergen inhalation challenge com-pared to pre-challenge, as well as lower in asthmaticscompared to healthy control subjects, suggesting thatthe change in miR-192 level may be involved in theatopic asthma and asthmatic reaction after allergen in-halation challenge. Cell specific expression of miR-192was associated with peripheral blood mononuclear cells(PBMCs). Among the genes we have previously reportedto be altered following allergen inhalation challenge [2],predicted target genes of miR-192 were largely up-regulated suggesting the inhibitory role of miR-192 ontheir expression. The result showing that these targetgenes were enriched for the functions on cell cycle andimmune response supports the notion that miRNA canregulate such biological functions in the asthmaticresponse.MiR-192 has been studied in various conditions in-cluding cancer and autoimmune diseases. Several reportsshowed that miR-192 affects cellular proliferationthrough the p53 pathway, which regulates cell cycle. Thecell cycle checkpoint control genes, p53 and p21 wereoverexpressed in cells with overexpressed miR-192in vitro using human cell lines [9]. In the canonical path-way modeling in our results for the up-regulated targetgenes for miR-192, cell cycle regulation and response toDNA damage was one of the top-listed pathways, sug-gesting that miR-192 mediates cell cycle regulation ofblood cells in response to allergen challenge. In theother study investigating the response of miRNA to en-vironment, exposure to cigarette smoke decreased miR-192 expression in the lung in animal experimental model[10]. Given that the cigarette smoke exposure inducesairway inflammation and cellular stress such as oxidativestress, this report supports our findings that miR-192 isregulating the response to miRNAs to environmental ex-posure inducing airway inflammation. As shown in theother top-listed pathway in the canonical pathway mod-eling in our data, miR-192 was suggested to mediate theimmune response following allergen inhalation. Inaddition, as a biomarker in peripheral blood, miR-192has been reported to decrease in systemic lupus erythe-matosus, a systemic autoimmune disease inducing in-flammatory responses [11]. Perturbation of miRNAprofiles in response to inflammatory stimuli can occurin peripheral blood and the changes can be detected.Interestingly, miR-192 expression is also reportedlyHC Pre Post HC Prep = 0.036Relative miR-192 expressionMean cellular miR-192 expressionA BFigure 3 Technical validation of miR-192 expression in whole blood and PBMCs. A. MiR-192 expression quantified using RT-qPCR in wholeblood. B. MiR-192 expression in PBMCs comparing healthy control (HC) and asthmatics (pre-challenge).Yamamoto et al. BMC Genomics 2012, 13:655 Page 5 of 9http://www.biomedcentral.com/1471-2164/13/655decreased in response to TGF-β and loss of miR-192correlates with tubulointerstitial fibrosis and reductionin renal function in renal biopsies from patients withestablished diabetic nephropathy [12]. Allergen inhal-ation challenge induces up-regulation of TGF-β in theairway epithelium [13]. TGF-β has been implicated inairway remodeling and inflammation, which are featuresof chronic asthma. Although the origin of the miRNAneeds to be clarified, our data showing down-regulatedmiR-192 in the blood after allergen inhalation challengemay indicate similar TGF-β derived mechanisms. Sincethe mechanism of miR-192 has not been elucidated inallergic airway diseases, further studies are needed toclarify these mechanisms.Peripheral blood consists of various types of cells suchthat the expression of a given miRNA is the net expres-sion from all the various cell-types. In this report, statis-tical methods for deconvolving cell-specific miRNAlevels from whole blood experiments were used. This ap-proach called cell-specific significance analysis of micro-arrays (csSAM) [14] can combine information fromcomplete blood cell count, including leukocyte differen-tials, and whole blood gene expression data to decon-volve cell specific expression measurements that canthen be compared across groups. We have previouslyshown that csSAM can uncover cell-specific gene ex-pression signatures in whole blood in two independentstudies [15,16]. We utilized this approach to analysecell-specific analysis for miR-192 expression in granulo-cytes and PBMCs. The difference of miR-192 levels issuggested to be derived from PBMCs in our data, sug-gesting that further studies on the subtype of lympho-cytes and monocytes will help reveal the mechanisms ofmiRNA in asthma and asthmatic responses as well.MiR-192 levels are reportedly different among subsets oflymphocytes [17]. A study comparing miRNA expressionin a wide range of haematological cell lines showed thatmiR-192 was up-regulated in activated B cells [18]. Al-lergen challenge induces a dynamic shift of lymphocytepopulations in blood. 24 hours after challenge there wasa reduction of peripheral blood CD4+ T lymphocytesfrom a baseline whereas CD4+ T lymphocytes in bronch-oalveolar fluid increased, suggesting lymphocyte recruit-ment into the respiratory system after allergen challenge[19,20]. In a baseline comparison between asthmaticsand healthy controls, while there was no significant dif-ference in number of T cells or B cells in peripheralblood [21,22], their subpopulations and activation statehave been reported to be different. Several studies inves-tigating peripheral blood cells in mild asthma alsoshowed the increase in the number of activated popula-tion such as CD23-bearing B cells and CD25-bearing Tcells in mild atopic asthmatics as well as low number ofgamma delta T cells in the peripheral blood ofasthmatics [22,23]. Given that cell populations and sub-populations can affect the miRNA levels, it is necessaryto clarify the relationship between such cellular popula-tions and differential miRNA profiles, for examplethrough the use of statistical deconvolution methodssuch as csSAM.The csSAM test statistic which compares the meanmiRNA expression for each cell type between two inde-pendent groups does not take into account the pairedstructure of our longitudinal study design. Since it is pos-sible to achieve statistical significance with smaller treat-ment effects in a paired study design, using an unpairedtest statistic may explain why the reduction of miR-192 ex-pression in PBMCs post-challenge compared to pre-challenge was not statistically significant (Figure 2B).Accounting for the within individual variation through theuse of a mixed-effects model or multilevel data analysiswith a modification to the csSAM test statistic may helpimprove the statistical significance of the csSAM test statis-tic in longitudinal studies. The small number of subjectslimited the number of cell-types for cell-specific analyses inthis study. Future studies with larger sample size will enableus to further study the role of additional cell types. Inaddition we have not compared the phenotypes induced byallergen inhalation challenge such as early responders anddual responders. The current analyses comparing pre andpost combined these phenotypes, which may serve as con-founding factors. Given the sample size a technical valid-ation with RT-qPCR was deemed appropriate. Given thecourse of allergen inhalation challenge, a longer timecourse study of miRNA levels in blood in both asth-matics and healthy subjects also needs to be explored.Collectively, a further study using a different cohort con-sisting of a large sample size is needed to validate the de-crease in miR-192 levels in asthmatics after allergeninhalation challenge. Thereafter, cell-specific approachesfor differential miRNAs will also validate our results aswell as shed light on further mechanisms of miRNAs incertain cell types in peripheral blood cells.ConclusionMiRNA profile changes can be detected in peripheralblood of asthmatic subjects undergoing allergen inhal-ation challenge and between healthy control and asth-matics. Among them, changes in miR-192 level may beinvolved in asthma mechanisms. These results indicatethat allergen inhalation challenge can be a suitablemodel to explore miRNA profiles and help elucidate themechanisms of allergic asthma in humans.MethodsSubjects and allergen inhalation challengeThis study was approved by University of BritishColumbia-Providence Health Care Research Ethics BoardYamamoto et al. BMC Genomics 2012, 13:655 Page 6 of 9http://www.biomedcentral.com/1471-2164/13/655with informed consents obtained in compliance with thelocal Research Ethics Boards. Seven subjects with stable,mild atopic asthma participated in the allergen inhalationchallenge. Four HCs were recruited to serve as controls. Allsubjects were non-smokers, free of other lung diseases, andnot pregnant. Diagnosis of asthma was based on the GlobalInitiative for Asthma criteria. Asthmatic subjects were diag-nosed as mild allergic asthma, and only used intermittentshort-acting bronchodilators for treatment of their asthma.Asthmatic subjects had a baseline FEV1 ≥70% of predicted,and the provocative concentration of methacholinerequired to produce a 20% decrease in FEV1 (PC20)was ≤16 mg/mL [24]. The methacholine inhalation chal-lenge was conducted as described by Cockcroft [25,26].Skin prick tests were used to determine allergies to cat, andthe dose of cat extract for inhalation. Allergen challengeswere conducted as triad visits. On the first and third days,subjects underwent methacholine challenges for assess-ments of airway hyperresponsiveness, and on the secondday subjects underwent allergen inhalation challenges asdescribed by O’ Byrne et al. [27], using extracts of cat peltor hair. Asthmatic subjects were challenged with cat aller-gen to reduce confounding effects of different types of aller-gens. Blood was drawn immediately before (pre) andapproximately 2 hours after allergen inhalation (post). Per-ipheral blood from HCs was collected in the morning(9 a.m.) to accommodate the time point comparing to pre-level of asthmatic subjects.Blood collection and RNA extractionPeripheral venous blood samples were collected into BDVacutainer plastic EDTA tubes (Becton, Dickinson andCompany, Franklin Lakes, NJ, USA). One aliquot wasprocessed for total and differential cell counts, and theother aliquot was frozen and stored at −80°C until RNAextraction. From thawed samples, total RNA containingmiRNA was purified from 400 μL of whole bloodaccording to manufacturer’s protocols using the RNeasyMini Kit (Qiagen, Chatsworth, CA, USA). The yield andquality of RNA were assessed by NanoDrop 8000 Spec-trophotometer (Thermo Scientific, Wilmington, DE,USA) and Agilent 2100 Bioanalyzer (Agilent Technolo-gies, Santa Clara, CA, USA).NanoString nCounter assayComprehensive assay for miRNA expression was per-formed using nCounterW miRNA Expression Assay Kits(NanoString Technologies, Seattle, WA, USA) at Nano-String Technologies. In this method, the novel technol-ogy which enabled multiplexed direct digital counting ofRNA molecules [28] were applied to miRNAs with somemodification. Briefly, to prepare a miRNA molecule forhybridization in the nCounter assay, a proprietary DNAsequence called miRtag is ligated to the mature miRNAusing a bridging oligonucleotide (bridge). The miRtagsfor the human miRNA are ligated and bridges are puri-fied in one simple multiplexed reaction. After removal ofthe bridge, the tagged mature miRNA is then hybridizedto a colour-coded reporter probe and a biotinylated cap-ture probe. The capture probe allows the complex to beimmobilized for data collection with measurement of itscolour code. A total of 734 human and human-associated viral miRNAs were simultaneously assayed.NanoString nCounter miRNA assay protocolMiRNA assays were performed using 100 ng of totalRNA following the standard nCounter miRNA AssayProtocol. Hybridizations were carried out by mixing 5 μlof each miRNA assay with 20 μl NanoString nCounterreporter probe and 5 μl capture probe (30 μl total reac-tion volume) and incubating the hybridizations at 65°Cfor 18 hours.Preprocessing of miRNA Panel CodesetThe nCounter assay for each sample consisted of sixpositive controls (0.125-128 fM), eight negative controls,five control mRNAs (ACTB, B2M, GAPDH, RPL19 andRPLP0) and 734 miRNAs. Prior to normalization severalprobes in the codeset required background subtraction(Additional file 1: Table S3). The probes for which thebackground subtraction calculation produced a negativenumber were set to 1 for simplicity. To account forslight differences in assay efficiency (hybridization, puri-fication, and binding) the data was normalized to thesum of 6 positive RNA spike-in controls. For each sam-ple, the mean plus 2 times the standard deviation of the8 negative controls was subtracted from each miRNAcount in that sample. Only miRNAs with non-negativecounts across all samples were retained for downstreamanalysis.Technical validation using RT-qPCRRT-qPCR was carried out using the TaqMan MiRNAAssay (Applied Biosystems, Foster City, CA) accordingto the manufacturer's protocol. RNA samples were mea-sured in duplicates. The TaqMan MicroRNA ReverseTranscription Kit (Applied Biosystems) was used for thepreparation of cDNA. Reverse transcription reactionswere performed in a volume of 15 μl, and each reactioncontained 10 ng of total RNA including miRNA. ThePCR reaction mix consisted of the RT product, TaqMan2X Universal PCR Master Mix and the appropriate 5XMicroRNA Assay Mix containing primers and probe forthe miRNA of interest. All TaqMan assays were run induplicate on an ABI Prism 7900, applying 40 PCR cycles.Ct values were calculated with the SDS software usingYamamoto et al. BMC Genomics 2012, 13:655 Page 7 of 9http://www.biomedcentral.com/1471-2164/13/655automatic baseline settings. Ct values >35 were consid-ered to be below the detection level of the assay. RNU44and RNU6B were used for normalizing the expressionlevel of selected miRNAs. The mean of Ct values wassubtracted from the corresponding Ct value for theselected miRNAs resulting in the ΔCt value which wasused for relative quantification of miRNA expression.Statistical analysisComplete blood cell count and leukocyte differentialswere compared among groups using analysis of variance.Moderated robust regression in the Linear Model forMicroarrays (limma) library from bioconductor was usedto determine statistically significant miRNAs using aBenjamini Hochberg FDR of 1% [29]. A p-value of 0.05was used to determine significant changes in cell counts.Significant cell-specific miRNA expression was deter-mined using methods as previously described [14]. Foreach group, regression of miRNA expression onto therelative cell-type frequencies was used to determine themean miRNA expression for each cell-type:1. Multiple regression of miRNA expression onto therelative cell-type frequencies for each group g.yg ¼ β0 þ β1gx1g þ    þ βkgxkg þ εg ¼ Xβþ ε;β0g = 0; implies that at zero cell-type frequency thereis zero miRNA expression.βkg; increase in miRNA expression for 1% increase inthe kth cell-type frequency in group g (mean miRNAexpression in the kth cell-type in group g)yg; vector of miRNA expression values for group gxkg: vector of relative cell-type frequencies for the kthcell-type for group g2. Test statistic to determine significant changes in cell-specific miRNA expression.Similar to the Wald test to determine significantcovariates, the following test statistic was used todetermine significant miRNA expression changes inthe kth cell between two groups.Test statistic comparing group 1 and group 2 for thekth cell-type:tk21 ¼ β^k2  β^k1se β^k 21;se β^k 21¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffin1 se β^k1 h i2þ n2 se β^k2 h i2n1 þ n2vuutThe p-value for each test-statistic was calculated bygenerating an empirical distribution by recalculatingtest-statistics after reshuffling of class labels 1000times. A p-value of 0.05 was deemed significant.Target prediction, gene ontology analysis and canonicalpathway analysisTarget genes for miR-192 were predicted using data-bases, miRanda and TargetScan to list the targets identi-fied by both. Then the target genes for miR-192 wereselected out of 1595 differentially expressed genes, whichwere identified post-allergen inhalation challenge at anFDR of 5% by Kam et al. [2].Signalling pathways and cellular processes for targetgenes, which were reportedly up-regulated in post-allergen challenge and predicted to be targeted by miR-192, were defined through GeneGo MetaCore databases:Functional Enrichment by Ontology and CanonicalPathway Modeling.Additional fileAdditional file 1: Supplementary Tables and Figures.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsGMG, PMO, CC, JMF, LPB, SJT participated in research design and provisionof samples. MY, JR, LPB, SJT participated in the sample processing andfollowing experiments. MY, AS, SJT conducted data analyses. MY, AS, CC, SJTparticipated in the writing of the paper. All authors read and approved thefinal manuscript.Availability of supporting dataSupplementary Tables and Figures are shown in Additional_file_1.pdf.AcknowledgementsThe authors wish to thank the research participants for taking part in thesestudies, as well as Johane Lepage, Philippe Prince, Joanne Milot, MylèneBertrand, Richard Watson, George Obminski, Heather Campbell, Abbey Torek,Tara Strinich and Karen Howie for their expertise and assistance with subjectrecruitment, allergen challenge and sample collection, as part of theAllerGen NCE Clinical Investigator Collaborative. This research was supportedby funding from AllerGen NCE Inc. (Allergy, Genes and EnvironmentNetwork) and the Canadian Institutes of Health Research. MY was supportedin part by the fellowship grants of a Canadian Institutes of Health Research(CIHR) Integrated and Mentored Pulmonary and Cardiovascular TrainingProgram (IMPACT), the Sumitomo Life Social Welfare Services Foundationand the Mochida Memorial Foundation for Medical and PharmaceuticalResearch.Author details1UBC James Hogg Research Centre, St. Paul’s Hospital, University of BritishColumbia, Room 166, Burrard Building, 1081 Burrard Street, Vancouver, BCV6Z 1Y6, Canada. 2Institute for HEART+LUNG Health, Vancouver, BritishColumbia, Canada. 3Department of Medicine, Division of RespiratoryMedicine, UBC, Vancouver, British Columbia, Canada. 4Vancouver CoastalHealth Research Institute, Vancouver General Hospital, Vancouver, BritishColumbia, Canada. 5NCE CECR PROOF Centre of Excellence, Vancouver,British Columbia, Canada. 6Department of Medicine, McMaster University,Hamilton, Ontario, Canada. 7Centre de Pneumologie de L’Hopital, UniversitéLaval, Sainte-Foy, Quebec, Canada.Received: 15 October 2012 Accepted: 15 November 2012Published: 21 November 2012References1. Gauvreau GM, Evans MY: Allergen inhalation challenge: a human modelof asthma exacerbation. Contrib Microbiol 2007, 14:21–32.Yamamoto et al. BMC Genomics 2012, 13:655 Page 8 of 9http://www.biomedcentral.com/1471-2164/13/6552. Kam SH, Singh A, He JQ, Ruan J, Gauvreau GM, O'Byrne PM, Fitzgerald JM,Tebbutt SJ: Peripheral blood gene expression changes during allergeninhalation challenge in atopic asthmatic individuals. J Asthma 2012, 49(3):219–226.3. Chaussabel D, Pascual V, Banchereau J: Assessing the human immunesystem through blood transcriptomics. BMC Biol 2010, 8:84.4. Esteller M: Non-coding RNAs in human disease. Nat Rev Genet 2011, 12(12):861–874.5. Garbacki N, Di Valentin E, Huynh-Thu VA, Geurts P, Irrthum A, Crahay C,Arnould T, Deroanne C, Piette J, Cataldo D, et al: MicroRNAs profiling inmurine models of acute and chronic asthma: a relationship with mRNAstargets. PLoS One 2011, 6(1):e16509.6. Lu TX, Munitz A, Rothenberg ME: MicroRNA-21 is up-regulated in allergicairway inflammation and regulates IL-12p35 expression. J Immunol 2009,182(8):4994–5002.7. Williams AE, Larner-Svensson H, Perry MM, Campbell GA, Herrick SE,Adcock IM, Erjefalt JS, Chung KF, Lindsay MA: MicroRNA expressionprofiling in mild asthmatic human airways and effect of corticosteroidtherapy. PLoS One 2009, 4(6):e5889.8. Jardim MJ, Dailey L, Silbajoris R, Diaz-Sanchez D: Distinct MicroRNAexpression in human airway cells of asthmatic donors identifies a novelasthma-associated gene. Am J Respir Cell Mol Biol 2012, 47(4):536–542.9. Song B, Wang Y, Kudo K, Gavin EJ, Xi Y, Ju J: miR-192 Regulatesdihydrofolate reductase and cellular proliferation through thep53-microRNA circuit. Clin Cancer Res 2008, 14(24):8080–8086.10. Izzotti A, Calin GA, Arrigo P, Steele VE, Croce CM, De Flora S:Downregulation of microRNA expression in the lungs of rats exposed tocigarette smoke. FASEB J 2009, 23(3):806–812.11. Wang G, Tam LS, Li EK, Kwan BC, Chow KM, Luk CC, Li PK, Szeto CC: Serumand urinary free microRNA level in patients with systemic lupuserythematosus. Lupus 2011, 20(5):493–500.12. Krupa A, Jenkins R, Luo DD, Lewis A, Phillips A, Fraser D: Loss ofMicroRNA-192 promotes fibrogenesis in diabetic nephropathy. J Am SocNephrol 2010, 21(3):438–447.13. Torrego A, Hew M, Oates T, Sukkar M, Fan Chung K: Expression andactivation of TGF-beta isoforms in acute allergen-induced remodelling inasthma. Thorax 2007, 62(4):307–313.14. Shen-Orr SS, Tibshirani R, Khatri P, Bodian DL, Staedtler F, Perry NM, Hastie T,Sarwal MM, Davis MM, Butte AJ: Cell type-specific gene expressiondifferences in complex tissues. Nat Methods 2010, 7(4):287–289.15. Shannon CP, Hollander Z, Wilson-McManus J, Balshaw R, Ng RT, McMaster R,McManus BM, Keown PA, Tebbutt SJ: White blood cell differentials enrichwhole blood expression data in the context of acute cardiac allograftrejection. Bioinform Biol Insights 2012, 6:49–61.16. Tebbutt SJ, He JQ, Singh A, Shannon CP, Ruan J, Carlsten C: Transcriptionalchanges of blood eosinophils after methacholine inhalation challenge inasthmatics. Genomics Insights 2012, 5:1–12.17. Rossi RL, Rossetti G, Wenandy L, Curti S, Ripamonti A, Bonnal RJ, Birolo RS,Moro M, Crosti MC, Gruarin P, et al: Distinct microRNA signatures inhuman lymphocyte subsets and enforcement of the naive state in CD4+T cells by the microRNA miR-125b. Nat Immunol 2011, 12(8):796–803.18. Lawrie CH, Saunders NJ, Soneji S, Palazzo S, Dunlop HM, Cooper CD,Brown PJ, Troussard X, Mossafa H, Enver T, et al: MicroRNA expressionin lymphocyte development and malignancy. Leukemia 2008,22(7):1440–1446.19. Gerblich AA, Campbell AE, Schuyler MR: Changes in T-lymphocytesubpopulations after antigenic bronchial provocation in asthmatics.N Engl J Med 1984, 310(21):1349–1352.20. Gerblich AA, Salik H, Schuyler MR: Dynamic T-cell changes in peripheralblood and bronchoalveolar lavage after antigen bronchoprovocation inasthmatics. Am Rev Respir Dis 1991, 143(3):533–537.21. Wilson JW, Djukanovic R, Howarth PH, Holgate ST: Lymphocyte activationin bronchoalveolar lavage and peripheral blood in atopic asthma. AmRev Respir Dis 1992, 145(4 Pt 1):958–960.22. Krejsek J, Kral B, Vokurkova D, Derner V, Touskova M, Parakova Z, Kopecky O:Decreased peripheral blood gamma delta T cells in patients withbronchial asthma. Allergy 1998, 53(1):73–77.23. Hallstrand TS, Ault KA, Bates PW, Mitchell J, Schoene RB: Peripheral bloodmanifestations of T(H)2 lymphocyte activation in stable atopic asthmaand during exercise-induced bronchospasm. Ann Allergy Asthma Immunol1998, 80(5):424–432.24. Gauvreau GM, Hessel EM, Boulet LP, Coffman RL, O'Byrne PM:Immunostimulatory sequences regulate interferon-inducible genes butnot allergic airway responses. Am J Respir Crit Care Med 2006, 174(1):15–20.25. Crapo RO, Casaburi R, Coates AL, Enright PL, Hankinson JL, Irvin CG,MacIntyre NR, McKay RT, Wanger JS, Anderson SD, et al: Guidelines formethacholine and exercise challenge testing-1999. This officialstatement of the American Thoracic Society was adopted by theATS Board of Directors, July 1999. Am J Respir Crit Care Med 2000,161(1):309–329.26. Cockcroft DW, Murdock KY, Kirby J, Hargreave F: Prediction of airwayresponsiveness to allergen from skin sensitivity to allergen and airwayresponsiveness to histamine. Am Rev Respir Dis 1987, 135(1):264–267.27. O'Byrne PM, Dolovich J, Hargreave FE: Late asthmatic responses. Am RevRespir Dis 1987, 136(3):740–751.28. Geiss GK, Bumgarner RE, Birditt B, Dahl T, Dowidar N, Dunaway DL, Fell HP,Ferree S, George RD, Grogan T, et al: Direct multiplexed measurement ofgene expression with color-coded probe pairs. Nat Biotechnol 2008,26(3):317–325.29. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B,Gautier L, Ge Y, Gentry J, et al: Bioconductor: open software developmentfor computational biology and bioinformatics. Genome Biol 2004,5(10):R80.doi:10.1186/1471-2164-13-655Cite this article as: Yamamoto et al.: Decreased miR-192 expression inperipheral blood of asthmatic individuals undergoing an allergeninhalation challenge. BMC Genomics 2012 13:655.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitYamamoto et al. BMC Genomics 2012, 13:655 Page 9 of 9http://www.biomedcentral.com/1471-2164/13/655


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