Evaluating normalization approaches for the better identification of aberrant microRNAs associated with hepatocellular carcinoma

Abstract

Aim: Dysregulated microRNAs (miRNAs) have been identified in hepatocellular carcinoma (HCC), but only a small proportion have been confirmed. An appropriate normalizer is crucial to determining the accuracy and reliability of data from miRNA studies.

Methods: Different normalization strategies were used to validate genome-wide miRNA profiles in HCC tumor and non-tumor tissues, and to determine the consistency and discrepancy of data on dysregulated miRNAs.

Results: Two sets of stable miRNAs (miR-30c/miR-30b and miR-30c/miR-126) were identified in HCC tissues by geNorm and NormFinder tools, respectively. The mean of global miRNAs also showed good stability for ranking the top 1-2 miRNAs, but the stabilities of the manufacturer-recommended ncRNAs controls were poor. Four panels of miRNAs were significantly associated with HCC by separately using various normalizers, and 14 miRNAs were consistently identified by three normalization strategies. Although fewer miRNAs (17-26) were dysregulated in HCC using the global mean or the 2 stable miRNAs as normalizers, perfect clustering of tissues was also obtained with only 1 to 2 misclassifications, suggesting the efficiency of the miRNA panels. Using global mean as the normalizer, the authors identified 7 miRNAs, including 2 novel (miR-324-5p and miR-550) significantly upregulated in HCC that were omitted when using 3 endogenous controls as the normalizer.

Conclusion: An optimal normalization strategy to identify biologically important miRNAs in HCC tissue studies of miRNA may be the combination of global mean and 2 stable miRNAs. Selection of appropriate normalization strategies to adjust miRNAs levels is particularly important for epidemiological studies dealing with large data sets and covering multiple experimental batches.

Keywords

MicroRNA / stability / normalization / hepatocellular carcinoma / biomarkers

References

[1] Lu J,Miska EA,Lamb J,Sweet-Cordero A,Mak RH,Downing JR,Horvitz HR.MicroRNA expression profiles classify human cancers..Nature2005;435:834-8

[2] Croce CM.Causes and consequences of microRNA dysregulation in cancer..Nat Rev Genet2009;10:704-14 PMCID:PMC3467096

[3] Sidhu K,Pandey V.The "macro" world of microRNAs in hepatocellular carcinoma..Front Oncol2015;5:68 PMCID:PMC4373247

[4] Borel F,Jansen PL.Diagnostic and therapeutic potential of miRNA signatures in patients with hepatocellular carcinoma..J Hepatol2012;56:1371-83

[5] Van Keuren-Jensen KR,Courtright AL,Starr AP,Beecroft TA,Kiefer JA,Rakela J.microRNA changes in liver tissue associated with fibrosis progression in patients with hepatitis C..Liver Int2016;36:334-43 PMCID:PMC5049661

[6] Naito Y,Ochiya T.microRNAs and hepatitis B..Adv Exp Med Biol2015;888:389-99

[7] Valencia-Quintana R,Tenorio-Arvide MG,Montiel-González JM,Villalobos-Pietrini R,Flores-Márquez AR.The microRNAs as potential biomarkers for predicting the onset of aflatoxin exposure in human beings: a review..Front Microbiol2014;5:102 PMCID:PMC3957091

[8] Yang Z,Wang L.Emerging role of microRNAs in lipid metabolism..Acta Pharm Sin B2015;5:145-50 PMCID:PMC4629215

[9] Zarfeshani A,Sheppard AM.MicroRNA expression relating to dietary-induced liver steatosis and NASH..J Clin Med2015;4:1938-50 PMCID:PMC4663477

[10] Zhang S,Jiang X,Lin Y,Xie W.Dysregulated serum microRNA expression profile and potential biomarkers in hepatitis C virus-infected patients..Int J Med Sci2015;12:590-8 PMCID:PMC4532963

[11] Shen J,Wang Q,Siegel AB,Santella RM.Exploration of genome-wide circulating microRNA in hepatocellular carcinoma: MiR-483-5p as a potential biomarker..Cancer Epidemiol Biomarkers Prev2013;22:2364-73 PMCID:PMC3963823

[12] Zhang Z,Wang X,Yan Q,Che Y,Zhang J.Serum miR-483-5p as a potential biomarker to detect hepatocellular carcinoma..Hepatol Int2013;7:199-207

[13] Bronte F,Fanale D,Bronte E,Fiorentino E,Bazan V,Russo A.HepatomiRNoma: the proposal of a new network of targets for diagnosis, prognosis and therapy in hepatocellular carcinoma..Crit Rev Oncol Hematol2016;97:312-21

[14] Mizuguchi Y,Yoshida H.Dysregulated microRNAs in progression of hepatocellular carcinoma: a systematic review..Hepatol Res2016;46:391-406

[15] Boisen MK,Linnemann D,Jensen BV,Johansen JS.MicroRNA expression in formalin-fixed paraffin-embedded cancer tissue: identifying reference microRNAs and variability..BMC Cancer2015;15:1024 PMCID:PMC4696166

[16] Wang Y,Shi W,Rao Q,Liu J,Du Z.Profiles of differential expression of circulating microRNAs in hepatitis B virus-positive small hepatocellular carcinoma..Cancer Biomark2015;15:171-80

[17] Wong CM,Lee JM,Au SL.Sequential alterations of microRNA expression in hepatocellular carcinoma development and venous metastasis..Hepatology2012;55:1453-61

[18] Augello C,Caruso L,Maggioni M,Montorsi M,Roncalli M.MicroRNA profiling of hepatocarcinogenesis identifies C19MC cluster as a novel prognostic biomarker in hepatocellular carcinoma..Liver Int2012;32:772-82

[19] Wang W,Tan YX,Qi ZT.Identification of deregulated miRNAs and their targets in hepatitis B virus-associated hepatocellular carcinoma..World J Gastroenterol2012;18:5442-53 PMCID:PMC3471114

[20] Wen Y,Chen J,Xia Y,Jiang Y,Lu J,Han J,Shen H,Hu Z.Plasma miRNAs as early biomarkers for detecting hepatocellular carcinoma..Int J Cancer2015;137:1679-90

[21] Oksuz Z,Kaplan E,Tezcan S,Emekdas G,Altintas E.Serum microRNAs; miR-30c-5p, miR-223-3p, miR-302c-3p and miR-17-5p could be used as novel non-invasive biomarkers for HCV-positive cirrhosis and hepatocellular carcinoma..Mol Biol Rep2015;42:713-20

[22] Zhuang L,Wang P.Serum miR-128-2 serves as a prognostic marker for patients with hepatocellular carcinoma..PLoS One2015;10:e0117274 PMCID:PMC4313939

[23] Mestdagh P,De Weer A,Westermann F,Vandesompele J.A novel and universal method for microRNA RT-qPCR data normalization..Genome Biol2009;10:R64 PMCID:PMC2718498

[24] Liang Y,Wong L.Characterization of microRNA expression profiles in normal human tissues..BMC Genomics2007;8:166 PMCID:PMC1904203

[25] Chugh P.Potential pitfalls in microRNA profiling..Wiley Interdiscip Rev RNA2012;3:601-16 PMCID:PMC3597218

[26] Wang X,Cairns MJ.Optimal consistency in microRNA expression analysis using reference-gene-based normalization..Mol Biosyst2015;11:1235-40

[27] Lin L,Yu J,Zhou A.Serum miR-224 as a biomarker for detection of hepatocellular carcinoma at early stage..Clin Res Hepatol Gastroenterol2016;40:397-404

[28] Shen J,Zhang YJ,Kibriya MG,Ahsan H,Siegel AB,Santella RM.Exploring genome-wide DNA methylation profiles altered in hepatocellular carcinoma using Infinium HumanMethylation 450 BeadChips..Epigenetics2013;8:34-43 PMCID:PMC3549879

[29] Shen J,Sirosh I,Tycko B.Integrative epigenomic and genomic filtering for methylation markers in hepatocellular carcinomas..BMC Med Genomics2015;8:28 PMCID:PMC4460673

[30] Shen J,Siegel AB,Wang Q,Santella RM.Genome-wide expression of microRNAs is regulated by DNA methylation in hepatocarcinogenesis..Gastroenterol Res Pract2015;2015:230642

[31] Edge SB.The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM..Ann Surg Oncol2010;17:1471-4

[32] Vandesompele J,Pattyn F,Van Roy N,Speleman F.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes..Genome Biol2002;3:1-11

[33] Andersen CL,Orntoft TF.Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets..Cancer Res2004;64:5245-50

[34] Simon R,Li MC,Menenzes S.Analysis of gene expression data using BRB-ArrayTools..Cancer Inform2007;3:11-7 PMCID:PMC2675854

[35] Heberle H,da Silva FR,Minghim R.InteractiVenn: a web-based tool for the analysis of sets through Venn diagrams..BMC Bioinformatics2015;16:169 PMCID:PMC4455604

[36] Pritchard CC,Tewari M.MicroRNA profiling: approaches and considerations..Nat Rev Genet2012;13:358-69 PMCID:PMC4517822

[37] Li ZB,Li L,Jia M.MiR-21 and miR-183 can simultaneously target SOCS6 and modulate growth and invasion of hepatocellular carcinoma (HCC) cells..Eur Rev Med Pharmacol Sci2015;19:3208-17

[38] Thurnherr T,Lei Z,Rozen SG.Differentially expressed miRNAs in hepatocellular carcinoma target genes in the genetic information processing and metabolism pathways..Sci Rep2016;6:20065 PMCID:PMC4730185

[39] Huang CS,Cui H,Zhang L,Huang T.Increased expression of miR-21 predicts poor prognosis in patients with hepatocellular carcinoma..Int J Clin Exp Pathol2015;8:7234-8 PMCID:PMC4525954

[40] Shi KQ,Chen XJ,Wang YQ,Yang NB,Dong JZ,Chen YP.Hepatocellular carcinoma associated microRNA expression signature: integrated bioinformatics analysis, experimental validation and clinical significance..Oncotarget2015;6:25093-108 PMCID:PMC4694817

[41] Zhang ZZ,Wang DQ,Niu LW,Liang Z.Hepatitis B virus and hepatocellular carcinoma at the miRNA level..World J Gastroenterol2011;17:3353-8 PMCID:PMC3160541

[42] Yuan R,Zhao H,Gao L,Kou Z,He S,Hu H.Upregulated expression of miR-106a by DNA hypomethylation plays an oncogenic role in hepatocellular carcinoma..Tumour Biol2015;36:3093-100

[43] Zhang QH,Zheng RZ,Zhang Q,Tang ZH.Meta-analysis of microRNA-183 family expression in human cancer studies comparing cancer tissues with noncancerous tissues..Gene2013;527:26-32

[44] Wojcicka A,Kornasiewicz O,Maciag M,Kotlarek M,Koperski L,Krawczyk M.Next generation sequencing reveals microRNA isoforms in liver cirrhosis and hepatocellular carcinoma..Int J Biochem Cell Biol2014;53:208-17

[45] Li J,Xu C,Xing R,Qin Y,Zheng X.miR-183 inhibits TGF-beta1-induced apoptosis by downregulation of PDCD4 expression in human hepatocellular carcinoma cells..BMC Cancer2010;10:354 PMCID:PMC2909210

[46] Li J,Yu W,Luo J.Expression of serum miR-221 in human hepatocellular carcinoma and its prognostic significance..Biochem Biophys Res Commun2011;406:70-3

[47] He XX,Xu CR,Xiang GY,Dan ZL,Liao JZ.Bioinformatics analysis identifies miR-221 as a core regulator in hepatocellular carcinoma and its silencing suppresses tumor properties..Oncol Rep2014;32:1200-10

[48] Sohn W,Kang SH,Cho JY,Shim SG.Serum exosomal microRNAs as novel biomarkers for hepatocellular carcinoma..Exp Mol Med2015;47:e184 PMCID:PMC4650928

[49] Pineau P,McJunkin K,Battiston C,Mazzaferro V,Croce CM.miR-221 overexpression contributes to liver tumorigenesis..Proc Natl Acad Sci U S A2010;107:264-9 PMCID:PMC2806773

[50] Shen H,Tian Y,Wang H,Li Y.Upregulation of miR-362-3p modulates proliferation and anchorage-independent growth by directly targeting Tob2 in hepatocellular carcinoma..J Cell Biochem2015;116:1563-73

[51] Ni F,Cui H,Chen L,Guo C,Chen Z,Chen D,Wang S.MicroRNA-362-5p promotes tumor growth and metastasis by targeting CYLD in hepatocellular carcinoma..Cancer Lett2015;356:809-18

[52] Wylie D,Choudhary A.A novel mean-centering method for normalizing microRNA expression from high-throughput RT-qPCR data..BMC Res Notes2011;4:555 PMCID:PMC3267743

[53] El-Halawany MS,Zeeneldin AA,Tantawy M,Hamed I.Investigating the pretreatment miRNA expression patterns of advanced hepatocellular carcinoma patients in association with response to TACE treatment..Biomed Res Int2015;2015:649750

[54] Du C,Cao L,Gyabaah OA,Zhou L,Zheng S.MiR-126-3p suppresses tumor metastasis and angiogenesis of hepatocellular carcinoma by targeting LRP6 and PIK3R2..J Transl Med2014;12:259 PMCID:PMC4189615

[55] Ghosh A,Datta S,Das S,Gupta S,Chowdhury A,Mohapatra SK.Hepatic miR-126 is a potential plasma biomarker for detection of hepatitis B virus infected hepatocellular carcinoma..Int J Cancer2016;138:2732-44

[56] Ebrahimi F,Smith RA.miR-126 in human cancers: clinical roles and current perspectives..Exp Mol Pathol2014;96:98-107

[57] Yanokura M,Iida M,Umene K,Kobayashi Y,Aoki D.MicroRNAS in endometrial cancer: recent advances and potential clinical applications..EXCLI J2015;14:190-8 PMCID:PMC4614112

[58] Hu L,Long H,Wang X,Li Y,Deng Y.Integrative microRNA and gene profiling data analysis reveals novel biomarkers and mechanisms for lung cancer..Oncotarget2016;7:8441-54