Small RNA sequencing and differential expression of miRNAs in colorectal cancer

Kwan-Liang Lye, Shiran Mohd Sidik, Sabariah Abdul Rahman, Yoke-Kqueen Cheah


MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in various cellular processes. Many studies have shown that miRNAs are dysregulated in various types of cancer. In Malaysia, there are no published studies on miRNA expression profile for colorectal cancer, which is among the most common cancer here and other parts of the world. Next-generation sequencing platform was introduced in recent years and has revolutionized the biomedical research settings. In this study, we performed small RNA sequencing on Illumina HiSeq 2000 platform and found that 22 miRNAs were significantly differential expressed between cancer and normal tissues. Further validation on qRT-PCR on 5 of the miRNAs selected showed 3 of them were up-regulated (hsa-miR-106a, hsa-miR-135b, hsa-miR-21) while 2 were down-regulated (hsa-miR1, hsa-miR-504). These findings may lead to a simple profiling method to distinguish the risk of imdividuals in developing colorectal cancer.

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Jemal A, Bray F, Center MM, Ferlay J, et al. Global Cancer Statistics. CA: A Cancer Journal for Clinicians 2011;61(2): 69–90. doi: 10.3322/caac.20107.

Lim GCC and Halimah Y. Second Report of the National Cancer Registry. Cancer Incidence in Malaysia 2003. National Cancer Registry, Kuala Lumpur. 2004

Ambros V.The functions of animals microRNAs. Nature 2004; 431(7006): 350–355. doi: 10.1038/nature02871.

Harfe BD. MicroRNAs in vertebrate development. Current Opinion in Genetics and Development 2005; 15(4): 410–415. doi: 10.1016/j.gde.2005.06.012.

Calin GA, Groce CM. MicroRNA signatures in human cancers. Nat Rev Cancer 2006; 6(11): 857–866. doi: 10.1038/nrc1997.

Cullen BR. Transcription and processing of human microRNA precursors. Molecular Cell 2004; 16(6): 861–865. doi: 10.1016/j.molcel.2004.12.002.

Dennis Lo YM, Chiu RWK. Next-generation sequencing of plasma/serum DNA: An emerging research and molecular diagnostic tool. Clinical Chemistry 2009; 55(4): 607–608. doi: 10.1373/clinchem.2009.

Calin CA, Ferracin M, Cimmino A, Di Leva G, Shimizu M, et al. A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. The New England Journal of Medicine 2005; 353(17): 1793–1801. doi: 10.1056/NEJMoa050995.

Bottoni A, Piccin D, Tagliati F, Luchin A, Zatelli MC, et al. miR-15a and miR-16-1 down-regulation in pituitary adenomas. Journal of Cellular Physiology 2005; 204(1): 280–285. doi: 10.1002/jcp.20282.

Cummins JM, He Y, Leary RJ, Pagliarini R, Diaz A, et al. The colorectal microRNAome. Proceedings of the National Academy of Sciences 2006; 103(10): 3687–3692. doi: 10.1073/pnas.0511155103.

Xi Y, Formentini A, Chien M, Weir DB, Russo JJ, et al. Prognostic values of microRNAs in colorectal cancer. Biomarker Insights 2006; 1: 113–121.

Dong Y, Wu WK, Wu CW, Sung JJ, Yu J, et al. MicroRNA dysregulation in colorectal cancer: a clinical perspective. Br J Cancer 2011; 104(6): 893–898. doi: 10.1038/bjc.2011.57.

Sarver AL, French AJ, Borralho PM, Thayanithy V, Oberg AL, et al. Human colon cancer profiles show differential microRNA expression depending on mismatch repair status and are characteristic of undifferentiated proliferative states. BMC Cancer 2009; 9: 401. doi: 10.1186/1471-2407-9-401.

Rao P K, Missiaglia E, Shields L, Greg Hyde G, Yuan B, et al. Distinct roles for miR-1 and miR-133a in the proliferation and differentiation of rhabdomyosarcoma cells. The FASEB Journal 2010; 24(9): 3427–3437. doi: 10.1096/fj.09-150698.

Nasser M W, Datta J, Nuovo G, Kutay H, Motiwala T, et al. Down-regulation of microRNA-1 (miR-1) in lung cancer. Journal of Biological Chemistry 2008; 283(48): 33394–33405. doi: 10.1074/jbc.M804788200.

Leone V, D’Angelo D, Rubio I, de Freitas PM, Federico A, et al. MiR-1 is a tumor suppressor in thyroid carcinogenesis targeting CCND2, CXCR4 and SDF-1. The Journal of Clinical Endocrinology & Metabolism 2011; 96(9): 1388–1398. doi: 10.1210/jc.2011-0345.

Hamfjord J, Stangeland AM, Hughes T, Skrede ML, Tveit KM, et al. Differential expression of miRNAs in colorectal cancer: comparison of paired tumor tissue and adjacent normal mucosa using high throughput sequencing. PLOS ONE 2012 7(4): e314150. doi: 10.1371/journal.pone.0034150.

Hu W, Chan CS, Wu R, Zhang C, Sun Y, et al. Negative regulation of tumor suppressor p53 by microRNA miR-504. Molecular Cell 2010; 38(5): 689–699. doi: 10.1016/j.molcel.2010.05.027.

Zhao L, Tang M, Hu Z, Yan B, Pi W, et al. miR-504 mediated down-regulation of nuclear respiratory factor 1 leads to radio-resistance in nasopharyngeal carcinoma. Oncotarget 2015; 6(18): 15995–-16018. doi: 10.18632/oncotarget.4138.

Jiang B, Gu Y, Chen Y. Identification of novel predictive markers for the prognosis of pancreatic ductal adenocarcinoma. Cancer Investigation 2014; 32(6): 218–225. doi: 10.3109/07357907.2014.905586.

Kikkawa N, Kinoshita T, Nohata N, Hanazawa T, Yamamoto N, et al. microRNA-504 inhibits cancer cell proliferation via targeting CDK6 in hypopharyngeal squamous cell carcinoma. International Journal of Oncology 2014; 44(6): 2085–2092. doi: 10.3892/ijo.2014.2349.

Guan Y, Chen L, Bao Y, Pang C, Cui R, et al. Downregulation of miR-504 is associated with poor prognosis in high-grade glioma. Int J Clin Exp Pathol 2015; 8(1): 727–734.

Cui R, Guan Y, Sun C, Wang Y. A tumor-suppresive microRNA, miR-504, inhibits cell proliferation and promotes apoptosis by targeting FOXP1 in human glioma. Cancer Letters 2016; 374(1): 1–11. doi: 10.1016/j.canlet.2016.01.051.

Slaby O, Svoboda M, Michalek J, Vyzula R. MicroRNAs in colorectal cancer: translation of molecular biology into clinical application. Molecular Cancer 2009; 8(102). doi:10.1186/1476-4598-8-102.

Agostini M, Pucciarelli S, Calore F, Bedin C, Enzo MV, et al. MiRNAs in colon and rectal cancer: A consensus for their true clinical value. Clinica Chimica Acta 2010; 411: 1181-1186. doi: 10.1016/j.cca.2010.05.002.

Srinivasan S, Patric IRP, Somasundaram K. A ten-microRNA expression signature predicts survival in glioblastoma. PLOS ONE 2011; 6(3): e17438. doi:10.1371/journal.pone.0017438.

Schetter AJ, Leung SY, Sohn JJ, Zanetti KA, Bowman ED, et al. MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. JAMA 2008; 299(4): 425–436. doi: 10.1001/jama.299.4.425.

Lum AM, Wang BB, Li L, Channa N, Bartha G, et al. Retroviral activation of the mir-106a microRNA cistron in T lymphoma. Retrovirology 2007; 4(5). doi:10.1186/1742-4690-4-5.

Yang G, Zhang R, Chen X, Mu Y, Ai J, et al. MiR-106a inhibits glioma cell growth by targeting E2F1 independent of p53 status. J Mol Med 2011; 89(10): 1037–1050. doi: 10.1007/s00109-011-0775-x.

McBee WC, Gardiner AS, Edwards RP, Lesnock JL, Bhargava R, et al. MicroRNA analysis in human papillomavirus (HPV)-associated cervical neoplasia and cancer. J Carcinogene Mutagene 2011; 2(1): 1–9. doi: 10.4172/2157-2518.1000114.

Bandres E, Cubedo E, Agirre X, Malumbres R, Zárate R, et al. Identification by real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues. Molecular Cancer 2006; 5(29). doi: 10.1186/1476-4598-5-29.

Nagel R, le Sage C, Diosdado B, van der Waal M, Oude Vrielink JA, et al. Regulation of the adenomatous polyposis coli gene by miR-135 family in colorectal cancer. Cancer Res 2008; 68(14): 5795–5802. doi: 10.1158/0008-5472.CAN-08-0951

Peralta-Zaragoza O, Deas J, Meneses-Acosta A, De la O-Gómez F, Fernández-Tilapa G, et al. Relevance of miR-21 in regulation of tumor suppressor gene PTEN in human cervical cancer cells. BMC Cancer 2016; 16(215). doi: 10.1186/s12885-016-2231-3.

Zhang X, Gee H, Rose B, Tran N. Regulation of the tumuor suppressor PDCD4 by miR-499 and miR-21 in oropharyngeal cancers. BMC Cancer 2016; 16(86). doi: 10.1186/s12885-016-2109-4.

Gu J-B, Bao X-B, Ma Z. Effects of miR-21 on proliferation and apoptosis in human gastric adenocarcinoma cells. Oncology Letters 2017. doi: 10.3892/ol.2017.6171.

Qi L, Bart J, Tan LP, Platteel I, Sluis Tv, et al. Expression of miR-21 and its targets (PTEN, PDCD4, TM1) in flat epithelial atypia of the breast in relation to ductal carcinoma in situ and invasive carcinoma. BMC Cancer 2009; 9(163). doi: 10.1186/1471-2407-9-163.

Asangani IA, Rasheed SA K, Nikolova DA, Leupold JH, Colburn NH, et al. MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 2008; 27(15)21288–2136. doi: 10.1038/sj.onc.1210856.

Wan P, Zou F, Zhang X, Li H, Dulak A, et al. microRNA-21 negatively regulates Cdc25A and cell cycle progression in colon cancer cells. Cancer Res 2009; 69(20): 8157–8165. doi: 10.1158/0008-5472.CAN-09-1996.

Seike M, Goto A, Okano T, Bowman ED, Schetter AJ, et al. MiR-21 is an EGFR-regulated anti-apoptotic factor in lung cancer in never-smokers. Proceedings of the National Academy of Sciences 2009; 106(29): 12085–12090. doi: 10.1073/pnas.0905234106.

Motameny S, Wolters S, Nurnberg P, Schumacher B. Next generation sequencing of miRNAs – strategies, resources and methods. Genes 2010; 1(1): 70–84. doi:10.3390/genes1010070.

Xiong M, Zhao Z, Arnold J, Yu F. Next-generation sequencing. Journal of Biomedicine and Biotechnology 2010. doi: 10.1155/2010/370710.

Chen X, Ba Y, Ma L, Cai X, Wang K, et al. Characterization of microRNAs in serum: A novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 2008; 18(10): 997–1006. doi: 10.1038/cr.2008.282.

Goh L, Chen GB, Cutcutach I, Low B, The BT, et al. Assessing matched normal and tumor pairs in next-generation sequencing studies. PLOS ONE 2011; 6(3): 1–5. doi: 10.1371/journal.pone.0017810.

Wu Q, Lu Z, Li H, Lu J, Guo L, et al. Next-generation sequencing of microRNAs for breast cancer detection. Journal of Biomedicine and Biotechnology 2011. doi: 10.1155/2011/597145.



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