Analyses of methylation status of CpG islands in promoters of miR-9 genes family in human gastric adenocarcinoma

Document Type : Original article


1 Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

2 Department of Genetics, School of Biological Sciences, Tarbiat Modares University, Tehran, Iran

3 Department of Hematology, Faculty of Medicine, Tarbiat Modares University, Tehran, Iran


In the recent years deregulation for microRNAs expression pattern have emerged as a possible molecular factor for carcinogenesis. It has been reported that the expression of miR-9 was down-regulated in human gastric adenocarcinoma. To figure out the molecular mechanism of this down regulation, the methylation status in promoters of miR-9 family loci were compared in the human gastric adenocarcinoma samples with their normal margins. Using a methylation specific PCR technique the methylation status of miR-9 family loci were compared between 30 pairs of primary human gastric adenocarcinoma samples with their normal margins. The methylation of miR 9-1 status showed no specific difference in promoter methylation pattern in tumor and normal specimens, while in the miR-9-2 locus were unmethylated in both types of tissues. The promoter of miR-9-3 locus seems to be specifically methylated in tumor and their normal margin tissues. Our data revealed methylation of these CpG islands were not meaningfully different between normal and tumor gastric adenocarcinoma specimens and the methylation status of promoter may not be able to account for alteration of miR-9 expression in this type of gastric cancer.


1. Smith MG, Hold GL, Tahara E, El-Omar EM. Cellular and molecular aspects of gastric cancer. World J Gastroenterol 2006;12:2979-2990.
2. Ueda T, Volinia S, Okumura H, Shimizu M, Taccioli C, Rossi S, Alder H, Liu CG, Oue N, Yasui W, Yoshida K, Sasaki H, Nomura S, Seto Y, Kaminishi M, Calin GA, Croce CM. Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis. Lancet Oncol. 2010 Feb;11(2):136-46.
3. Yao Y, Suo AL, Li ZF, Liu LY, Tian T, Ni L, Zhang WG, Nan KJ, Song TS, Huang C.MicroRNA profiling of human gastric cancer. Mol Med Rep 2009;2:963-970.
4. Takagi T, Iio A, Nakagawa Y, Naoe T, Tanigawa N, Akao Y. Decreased expression of microRNA-143 and -145 in human gastric cancers. Oncology 2009;77:12-21.
5. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, andfunction. Cell 2004; 116:281-297.
6. Brennecke J, Stark A, Russell RB, Cohen SM. Principles of microRNA-target recognition. PLoS Biol 2005;3:e85.
7. Esquela-Kerscher A, Slack FJ. OncomiRs-microRNAs with a role in cancer. Nat Rev  Cancer 2006;6:259-269.
8. Hammond SM. MicroRNAs as tumor suppressors. Nat Genet 2007;39:582-583.
9. Tavazoie SF, Alarcón C, Oskarsson T, Padua D, Wang Q, Bos PD, Gerald WL, Massagué J.  Endogenous human microRNAs that suppress breast cancer metastasis. Nature 2008;451:147-152.
10. He L, He X, Lim LP, de Stanchina E, Xuan Z, Liang Y, Xue W, Zender L, Magnus J, Ridzon D, Jackson AL, Linsley PS, Chen C, Lowe SW, Cleary MA, Hannon GJ.A micro-RNA component of the p53 tumor suppressor network. Nature 2007;447:1130-1134.
11. Saito Y, Jones PA. Epigenetic activation of tumor suppressor micro-RNAs in human cancer cells. Cell Cycle 2006;5:2220-2222.
12. Deng S, Calin GA, Croce CM, Coukos G, Zhang L. Mechanisms of microRNA deregulation in human cancer. Cell Cycle 2008;7:2643-2646.
13. Saito Y, Liang G, Egger G, Friedman JM, Chuang JC, Coetzee GA, Jones PA. Specific activation of microRNA-127 with down-regulation of the proto-oncogene BCL6 by chromatin-modifying drugs in human cancer cells. Cancer Cell 2006;9:435-443.
14. Scott GK, Mattie MD, Berger CE, Benz SC, Benz CC. Rapid alteration of microRNA levels by histone deacetylase inhibition. Cancer Res 2006;66:1277-1281.
15. Lujambio A, Calin GA, Villanueva A, Ropero S, Sánchez-Céspedes M, Blanco D, Montuenga LM, Rossi S, Nicoloso MS, Faller WJ, Gallagher WM, Eccles SA, Croce CM, Esteller M. A microRNA DNA methylation signature for human cancer metastasis. Proc Natl Acad Sci USA 2008;105:13556-13561.
16. Lehmann U, Hasemeier B, Christgen M, Müller M, Römermann D, Länger F, Kreipe H. Epigenetic inactivation of microRNA gene hsa-mir-9-1 in human breast cancer. J Pathol 2008;214:17-24.
17. Ma L, Young J, Prabhala H, Pan E, Mestdagh P, Muth D, Teruya-Feldstein J, Reinhardt F, Onder TT, Valastyan S, Westermann F, Speleman F, Vandesompele J, Weinberg RA. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat Cell Biol 2010;12:247-256.
18. Bandres E, Agirre X, Bitarte N, Ramirez N, Zarate R, Roman-Gomez J, Prosper F, Garcia-Foncillas J. Epigenetic regulation of microRNA expression in colorectal cancer. Int J Cancer 2009;125:2737-2743.
19. Luo H, Zhang H, Zhang Z, Zhang X, Ning B, Guo J, Nie N, Liu B, Wu X. Down-regulated miR-9 and miR-433 in human gastric carcinoma. J Exp Clin Cancer Res 2009;28:82.
20. Wan HY, Guo LM, Liu T, Liu M, Li X, Tang H. Regulation of the transcription factor NF-κB1 by microRNA-9 in human gastric adenocarcinoma.Mol Cancer 2010; 9:16.
21. Gramantieri L, Ferracin M, Fornari F, Veronese A, Sabbioni S, Liu CG, Calin GA, Giovannini C, Ferrazzi E, Grazi GL, Croce CM, Bolondi L, Negrini M. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer Res 2007;67:6092-6099.
22. He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S, Powers S, Cordon-Cardo C, Lowe SW, Hannon GJ, Hammond SM. A microRNA polycistron as a potential human oncogene. Nature 2005;435:828-833.
23. O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression.Nature 2005;435:839-843.
24. Subramanian S, Lui WO, Lee CH, Espinosa I, Nielsen TO, Heinrich MC, Corless CL, Fire AZ, van de Rijn M. MicroRNA expression signature of human sarcomas. Oncogene 2008;27:2015-2026.
25. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR. MicroRNA expression profiles classify human cancers. Nature 2005;435:834-838.
26. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA 2006;103:2257-2261.
27. Marson A, Levine SS, Cole MF, Frampton GM, Brambrink T, Johnstone S, Guenther  MG, Johnston WK, Wernig M, Newman J, Calabrese JM, Dennis LM, Volkert TL, Gupta S, Love J, Hannett N, Sharp PA, Bartel DP, Jaenisch R, Young RA. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell 2008;134:521-533.
28. Laios A, O'Toole S, Flavin R, Martin C, Kelly L, Ring M, Finn SP, Barrett C, Loda M, Gleeson N, D'Arcy T, McGuinness E, Sheils O, Sheppard B, O'Leary J. Potential role of miR-9 and miR-223 in recurrent ovarian cancer. Mol Cancer 2008;7:35.
29. Guo LM, Pu Y, Han Z, Liu T, Li YX, Liu M, Li X, Tang H. MicroRNA-9 inhibits ovarian cancer cell growth through regulation of NF-κB1. FEBS J 2009;276:5537-5546.
30. Ertel A, Verghese A, Byers SW, Ochs M, Tozeren A. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells. Mol Cancer 2006;5:55.
31. Kerkel K, Spadola A, Yuan E, Kosek J, Jiang L, Hod E, Li K, Murty VV, Schupf N, Vilain E, Morris M, Haghighi F, Tycko B. Genomic surveys by methylation-sensitive SNP analysis identify sequence-dependent allele-specific DNA methylation. Nat Genet 2008;40:904-908. 
32. Tycko B. Allele-specific DNA methylation: beyond imprinting. Hum Mol Genet 2010;19:R210-R220.
33. Hellman A, Chess A. Extensive sequence-influenced DNA methylation polymorphism in the human genome. Epigenetics Chromatin 2010;3:11.
34. Rotkrua P, Akiyama Y, Hashimoto Y, Otsubo T, Yuasa Y. MiR-9 down-regulates CDX2 expression in gastric cancer cells. Int J Cancer 2011;129:2611-2620.
35. Tsai KW, Liao YL, Wu CW, Hu LY, Li SC, Chan WC, Ho MR, Lai CH, Kao HW, Fang WL, Huang KH, Lin WC. Aberrant hypermethylation of miR-9 genes in gastric cancer. Epigenetics 2011;6:1189-1197.