Methods and matrices: approaches to identifying miRNAs for Nasopharyngeal carcinoma

Journal of Translational Medicine

Jason P Mulvenna³  Jeffrey M Bethony⁵  Paul J Brindley⁵  Nader Sadeghi²  Salman Hashmi¹  Elizabeth Martinez⁴  Samantha Easley⁴  Paul H Levine¹  Yanjung Feng⁵  Gabriel Rinaldi⁶  Jordan L Plieskatt⁵ 

[1] Department of Epidemiology and Biostatistics, The George Washington University School of Public Health and Health Services, Washington, DC 20037, USA;Medical Faculty Associates, The George Washington University, Washington, DC 20037, USA;Infectious Disease and Cancer, Queensland Institute for Medical Research, Brisbane, Australia;Department of Pathology, School of Medicine and Health Science, George Washington University, Washington, DC, USA;Research Center for Neglected Diseases of Poverty, School of Medicine and Health Science, George Washington University, Washington, DC, USA;Departamento de Genética, Facultad de Medicina, Universidad de la República, (UDELAR), Montevideo 11800, Uruguay

关键词: Tumor;    RNA-Seq;    Next generation sequencing;    qPCR;    MicroRNAs;    Biomarker;    Methods;    Nasopharyngeal carcinoma;   

Others  :  822341
DOI  :  10.1186/1479-5876-12-3

 received in 2013-09-06, accepted in 2013-12-22,  发布年份 2014

【 摘 要 】

Background

Nasopharyngeal carcinoma (NPC) is a solid tumor of the head and neck. Multimodal therapy is highly effective when NPC is detected early. However, due to the location of the tumor and the absence of clinical signs, early detection is difficult, making a biomarker for the early detection of NPC a priority. The dysregulation of small non-coding RNAs (miRNAs) during carcinogenesis is the focus of much current biomarker research. Herein, we examine several miRNA discovery methods using two sample matrices to identify circulating miRNAs (c-miRNAs) associated with NPC.

Methods

We tested two miRNA discovery workflows on two sample sources for miRNAs associated with NPC. In the first workflow, we assumed that NPC tumor tissue would be enriched for miRNAs, so we compared miRNA expression in FFPE from NPC cases and controls using microarray and RNA-Seq technologies. Candidate miRNAs from both technologies were verified by qPCR in FFPE and sera from an independent NPC sample set. In a second workflow, we directly interrogated NPC case and control sera by RNA-Seq for c-miRNAs associated with NPC, with candidate c-miRNAs verified by qPCR in the sera from the same independent NPC sample set.

Results

Both microarray and RNA-Seq narrowed the miRNA signature to 1-5% of the known mature human miRNAs. Moreover, these two methods produced similar results when applied to the same sample type (FFPE), with RNA-Seq additionally indicating “unknown” miRNAs associated with NPC. However, we found different miRNA profiles in NPC sera compared to FFPE using RNA-Seq, with the few overlapping miRNAs found to be significantly up-regulated in FFPE significantly down-regulated in sera (and vice versa). Despite the different miRNA profiles found in FFPE and sera, both profiles strongly associated with NPC, providing two potential sources for biomarker signatures for NPC.

Conclusions

We determined that the direct interrogation of sera by RNA-Seq was the most informative method for identifying a c-miRNA signature associated with NPC. We also showed that there are different miRNA expression profiles associated with NPC for tumor tissue and sera. These results reflect on the methods and meaning of miRNA biomarkers for NPC in tissue and peripheral blood.

【 授权许可】

   
2014 Plieskatt et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]McDermott AL, Dutt SN, Watkinson JC: The aetiology of nasopharyngeal carcinoma. Clin Otolaryngol Allied Sci 2001, 26:82-92.
• [2]Yu MC, Yuan JM: Epidemiology of nasopharyngeal carcinoma. Semin Cancer Biol 2002, 12:421-429.
• [3]Gourzones C, Barjon C, Busson P: Host-tumor interactions in nasopharyngeal carcinomas. Semin Cancer Biol 2012, 22:127-136.
• [4]Chang KP, Hao SP, Chang JH, Wu CC, Tsang NM, Lee YS, Hsu CL, Ueng SH, Liu SC, Liu YL, et al.: Macrophage inflammatory protein-3alpha is a novel serum marker for nasopharyngeal carcinoma detection and prediction of treatment outcomes. Clin Cancer Res: Off J Am Assoc Cancer Res 2008, 14:6979-6987.
• [5]Li J, Zeng XH, Mo HY, Rolen U, Gao YF, Zhang XS, Chen QY, Zhang L, Zeng MS, Li MZ, et al.: Functional inactivation of EBV-specific T-lymphocytes in nasopharyngeal carcinoma: implications for tumor immunotherapy. PloS One 2007, 2:e1122.
• [6]Gourzones C, Gelin A, Bombik I, Klibi J, Verillaud B, Guigay J, Lang P, Temam S, Schneider V, Amiel C, et al.: Extra-cellular release and blood diffusion of BART viral micro-RNAs produced by EBV-infected nasopharyngeal carcinoma cells. Virol J 2010, 7:271. BioMed Central Full Text
• [7]Lin JC, Wang WY, Chen KY, Wei YH, Liang WM, Jan JS, Jiang RS: Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma. New Engl J Med 2004, 350:2461-2470.
• [8]Lo KW, Lo YM, Leung SF, Tsang YS, Chan LY, Johnson PJ, Hjelm NM, Lee JC, Huang DP: Analysis of cell-free Epstein-Barr virus associated RNA in the plasma of patients with nasopharyngeal carcinoma. Clin Chem 1999, 45:1292-1294.
• [9]Mutirangura A, Pornthanakasem W, Theamboonlers A, Sriuranpong V, Lertsanguansinchi P, Yenrudi S, Voravud N, Supiyaphun P, Poovorawan Y: Epstein-Barr viral DNA in serum of patients with nasopharyngeal carcinoma. Clin Cancer Res: Off J Am Assoc Cancer Res 1998, 4:665-669.
• [10]Wong AM, Kong KL, Tsang JW, Kwong DL, Guan XY: Profiling of Epstein-Barr virus-encoded microRNAs in nasopharyngeal carcinoma reveals potential biomarkers and oncomirs. Cancer 2012, 118:698-710.
• [11]Fachiroh J, Stevens SJ, Haryana SM, Middeldorp JM: Combination of Epstein-Barr virus scaffold (BdRF1/VCA-p40) and small capsid protein (BFRF3/VCA-p18) into a single molecule for improved serodiagnosis of acute and malignant EBV-driven disease. J Virol Methods 2010, 169:79-86.
• [12]Houali K, Wang X, Shimizu Y, Djennaoui D, Nicholls J, Fiorini S, Bouguermouh A, Ooka T: A new diagnostic marker for secreted Epstein-Barr virus encoded LMP1 and BARF1 oncoproteins in the serum and saliva of patients with nasopharyngeal carcinoma. Clin Cancer Res: Off J Am Assoc Cancer Res 2007, 13:4993-5000.
• [13]Joab I, Nicolas JC, Schwaab G, de-The G, Clausse B, Perricaudet M, Zeng Y: Detection of anti-Epstein-Barr-virus transactivator (ZEBRA) antibodies in sera from patients with nasopharyngeal carcinoma. Int J Cancer 1991, 48:647-649.
• [14]Xu J, Ahmad A, Blagdon M, D'Addario M, Jones JF, Dolcetti R, Vaccher E, Prasad U, Menezes J: The Epstein-Barr virus (EBV) major envelope glycoprotein gp350/220-specific antibody reactivities in the sera of patients with different EBV-associated diseases. Int J Cancer 1998, 79:481-486.
• [15]Pow EH, Law MY, Tsang PC, Perera RA, Kwong DL: Salivary Epstein-Barr virus DNA level in patients with nasopharyngeal carcinoma following radiotherapy. Oral Oncol 2011, 47:879-882.
• [16]Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004, 116:281-297.
• [17]Cullen BR: Viruses and microRNAs. Nat Genet 2006, 38(Suppl):S25-S30.
• [18]He L, Hannon GJ: MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 2004, 5:522-531.
• [19]Huang X, Yuan T, Tschannen M, Sun Z, Jacob H, Du M, Liang M, Dittmar RL, Liu Y, Kohli M, et al.: Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genomics 2013, 14:319. BioMed Central Full Text
• [20]Chen SJ, Chen GH, Chen YH, Liu CY, Chang KP, Chang YS, Chen HC: Characterization of Epstein-Barr virus miRNAome in nasopharyngeal carcinoma by deep sequencing. PloS One 2010, 5:9.
• [21]Wittmann J, Jack HM: Serum microRNAs as powerful cancer biomarkers. Biochim Biophys Acta 1806, 2010:200-207.
• [22]Asaga S, Kuo C, Nguyen T, Terpenning M, Giuliano AE, Hoon DS: Direct serum assay for microRNA-21 concentrations in early and advanced breast cancer. Clin Chem 2011, 57:84-91.
• [23]Pu XX, Huang GL, Guo HQ, Guo CC, Li H, Ye S, Ling S, Jiang L, Tian Y, Lin TY: Circulating miR-221 directly amplified from plasma is a potential diagnostic and prognostic marker of colorectal cancer and is correlated with p53 expression. J Gastroenterol Hepatol 2010, 25:1674-1680.
• [24]Resnick KE, Alder H, Hagan JP, Richardson DL, Croce CM, Cohn DE: The detection of differentially expressed microRNAs from the serum of ovarian cancer patients using a novel real-time PCR platform. Gynecol Oncol 2009, 112:55-59.
• [25]Doleshal M, Magotra AA, Choudhury B, Cannon BD, Labourier E, Szafranska AE: Evaluation and validation of total RNA extraction methods for microRNA expression analyses in formalin-fixed, paraffin-embedded tissues. J Mol Diagn 2008, 10:203-211.
• [26]Kotorashvili A, Ramnauth A, Liu C, Lin J, Ye K, Kim R, Hazan R, Rohan T, Fineberg S, Loudig O: Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens. PloS One 2012, 7:e34683.
• [27]Liu A, Xu X: MicroRNA isolation from formalin-fixed, paraffin-embedded tissues. Methods Mol Biol 2011, 724:259-267.
• [28]Weng L, Wu X, Gao H, Mu B, Li X, Wang JH, Guo C, Jin JM, Chen Z, Covarrubias M, et al.: MicroRNA profiling of clear cell renal cell carcinoma by whole-genome small RNA deep sequencing of paired frozen and formalin-fixed, paraffin-embedded tissue specimens. J Pathol 2010, 222:41-51.
• [29]Ablashi DV, Prasad U, Pearson GR, Prathap K, Armstrong GR, Faggioni A, Yadav M, Easton JM, Chan SH, Levine PH: EBV related studies with clinicopathological correlation in Malaysian nasopharyngeal carcinoma. In Nasopharyngeal carcinoma: current concepts. Edited by Prasad U, Ablashi DV, Levine PH, Pearson GR. Kuala Lumpur: University of Malaya Press; 1983:163-171.
• [30]Anta L, Blanco JL, Llibre JM, Garcia F, Perez-Elias MJ, Aguilera A, Perez-Romero P, Caballero E, Vidal C, Canizares A, et al.: Resistance to the most recent protease and non-nucleoside reverse transcriptase inhibitors across HIV-1 non-B subtypes. J Antimicrob Chemother 2013, 68:1994-2002.
• [31]Bertram G, Pearson GR, Faggioni A, Krueger GRF, Sesterhenn K, Ablashi DV, Levine PH: A long term study of EBV and non EBV related tests and their correlation with the clinical course of nasopharyngeal carcinoma. In Nasopharyngeal carcinoma: current concepts. Edited by Prasad U, Ablashi DV, Levine PH. Pearson GR: University of Malaya Press; 1983:115-124.
• [32]Goodman ML, Levine PH, Miller D: Nasopharyngeal carcinoma patients in Massachusetts. In Third International symposium on prevention and detection of cancer [Part 1]. Edited by Nieburgs HE. New York: Marcel Dekker,Inc; 1978:1591-1598.
• [33]Levine PH, De The GB, Brugere G, Schwaab G, Mourali N, Herberman RB, Ambrosioni JC, Revol P: Immunity to antigens associated with a cell line derived from nasopharyngeal cancer (NPC) in non-Chinese NPC patients. Int J Cancer 1976, 17:155-160.
• [34]Levine PH, Wallen WC, Ablashi DV, Granlund DJ, Connelly R: Comparative studies on immunity to EBV-associated antigens in NPC patients in North America, Tunisia, France and Hong Kong. Int J Cancer 1977, 20:332-338.
• [35]Li G, Wu Z, Peng Y, Liu X, Lu J, Wang L, Pan Q, He ML, Li XP: MicroRNA-10b induced by Epstein-Barr virus-encoded latent membrane protein-1 promotes the metastasis of human nasopharyngeal carcinoma cells. Cancer Lett 2010, 299:29-36.
• [36]Liu N, Chen NY, Cui RX, Li WF, Li Y, Wei RR, Zhang MY, Sun Y, Huang BJ, Chen M, et al.: Prognostic value of a microRNA signature in nasopharyngeal carcinoma: a microRNA expression analysis. Lancet Oncol 2012, 13:633-641.
• [37]Shen J, Todd NW, Zhang H, Yu L, Lingxiao X, Mei Y, Guarnera M, Liao J, Chou A, Lu CL, et al.: Plasma microRNAs as potential biomarkers for non-small-cell lung cancer. Lab Invest; J Tech Methods Pathol 2011, 91:579-587.
• [38]Sita-Lumsden A, Dart DA, Waxman J, Bevan CL: Circulating microRNAs as potential new biomarkers for prostate cancer. Br J Cancer 2013, 108:1925-1930.
• [39]Edge S, Byrd D, Compton C, Fritz A, Greene F, Trotti A: AJCC cancer staging manual. 7th edition. New York, NY: Springer; 2010.
• [40]Chan J, Pilch B, Kuo T, Wenig B, Lee A: Nasopharyngeal carcinoma. Lyon, France: IARC Press; 2005.
• [41]Tarone RE, Levine PH, Yadav M, Pandey JP: Relationship between Immunoglobulin Allotypes and Susceptibility to Nasopharyngeal Carcinoma in Malaysia. Cancer Res 1990, 50:3186-3188.
• [42]Friedlander MR, Chen W, Adamidi C, Maaskola J, Einspanier R, Knespel S, Rajewsky N: Discovering microRNAs from deep sequencing data using miRDeep. Nat Biotechnol 2008, 26:407-415.
• [43]Wang WC, Lin FM, Chang WC, Lin KY, Huang HD, Lin NS: miRExpress: analyzing high-throughput sequencing data for profiling microRNA expression. BMC Bioinformatics 2009, 10:328. BioMed Central Full Text
• [44]Kozomara A, Griffiths-Jones S: miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res 2011, 39:D152-D157.
• [45]Robinson MD, Smyth GK: Moderated statistical tests for assessing differences in tag abundance. Bioinformatics 2007, 23:2881-2887.
• [46]Robinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010, 26:139-140.
• [47]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 2001, 25:402-408.
• [48]Xi Y, Nakajima G, Gavin E, Morris CG, Kudo K, Hayashi K, Ju J: Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA 2007, 13:1668-1674.
• [49]Jung M, Schaefer A, Steiner I, Kempkensteffen C, Stephan C, Erbersdobler A, Jung K: Robust microRNA stability in degraded RNA preparations from human tissue and cell samples. Clin Chem 2010, 56:998-1006.
• [50]Li T, Chen JX, Fu XP, Yang S, Zhang Z, Chen Kh H, Li Y: microRNA expression profiling of nasopharyngeal carcinoma. Oncol Rep 2011, 25:1353-1363.
• [51]Wong TS, Man OY, Tsang CM, Tsao SW, Tsang RK, Chan JY, Ho WK, Wei WI, To VS: MicroRNA let-7 suppresses nasopharyngeal carcinoma cells proliferation through downregulating c-Myc expression. J Cancer Res Clin Oncol 2011, 137:415-422.
• [52]Chen HC, Chen GH, Chen YH, Liao WL, Liu CY, Chang KP, Chang YS, Chen SJ: MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma. Br J Cancer 2009, 100:1002-1011.
• [53]Shi W, Alajez NM, Bastianutto C, Hui AB, Mocanu JD, Ito E, Busson P, Lo KW, Ng R, Waldron J, et al.: Significance of Plk1 regulation by miR-100 in human nasopharyngeal cancer. Int J Cancer 2010, 126:2036-2048.
• [54]Zhang L, Deng T, Li X, Liu H, Zhou H, Ma J, Wu M, Zhou M, Shen S, Li X, et al.: microRNA-141 is involved in a nasopharyngeal carcinoma-related genes network. Carcinogenesis 2010, 31:559-566.
• [55]Gourzones C, Jimenez AS, Busson P: Profiling of Epstein-Barr virus-encoded microRNAs in nasopharyngeal carcinoma reveals potential biomarkers and oncomirs. Cancer 2012, 118:4634. author reply 4634–4635
• [56]Git A, Dvinge H, Salmon-Divon M, Osborne M, Kutter C, Hadfield J, Bertone P, Caldas C: Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression. RNA 2010, 16:991-1006.
• [57]Wei WI, Sham JS: Nasopharyngeal carcinoma. Lancet 2005, 365:2041-2054.
• [58]Calin GA, Croce CM: MicroRNA signatures in human cancers. Nat Rev Cancer 2006, 6:857-866.
• [59]Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen A, et al.: Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 2008, 105:10513-10518.
• [60]Guo L, Zhao Y, Yang S, Cai M, Wu Q, Chen F: Genome-wide screen for aberrantly expressed miRNAs reveals miRNA profile signature in breast cancer. Mol Biol Rep 2013, 40:2175-2186.
• [61]Gallo A, Tandon M, Alevizos I, Illei GG: The majority of microRNAs detectable in serum and saliva is concentrated in exosomes. PloS One 2012, 7:e30679.
• [62]Meckes DG Jr, Shair KH, Marquitz AR, Kung CP, Edwards RH, Raab-Traub N: Human tumor virus utilizes exosomes for intercellular communication. Proc Natl Acad Sci USA 2010, 107:20370-20375.
• [63]Gourzones C, Ferrand FR, Amiel C, Verillaud B, Barat A, Guerin M, Gattolliat CH, Gelin A, Klibi J, Chaaben AB, et al.: Consistent high concentration of the viral microRNA BART17 in plasma samples from nasopharyngeal carcinoma patients–evidence of non-exosomal transport. Virol J 2013, 10:119. BioMed Central Full Text
• [64]Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL, et al.: Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci USA 2011, 108:5003-5008.
• [65]Eldh M, Lotvall J, Malmhall C, Ekstrom K: Importance of RNA isolation methods for analysis of exosomal RNA: evaluation of different methods. Mol Immunol 2012, 50:278-286.
• [66]Markou A, Sourvinou I, Vorkas PA, Yousef GM, Lianidou E: Clinical evaluation of microRNA expression profiling in non small cell lung cancer. Lung Cancer 2013, 81(3):388-396.