title: Epigenetic silencing of miRNA-9 is correlated with promoter-proximal CpG island hypermethylation in gastric cancer in vitro and in vivo.
abstract: Silencing of protein-coding tumor suppressor genes (TSGs) by CpG island hypermethylation is a common occurrence in gastric cancer (GC). Here, we examine if tumor suppressor microRNAs (miRNAs) are silenced in a similar manner. Real-time quantitative PCR (RTQ-PCR) was employed to investigate the expression level of four candidate miRNAs in GC tissues (n=30) and cell lines. Basing on RTQ-PCR results and bioinformatics approach, miR-9 was chosen for further study on epigenetic regulation. Bisulfite genomic sequencing PCR (BSP) was performed to assess the methylation status of miR-9 in GC tissues. In both GC cell lines and animal models, demethylation was performed either by treatment with 5-aza-2'-deoxycytidine (5-AZA-CdR) or by siRNA targeting DNMT1. We also analyzed the relationship between miRNAs and several clinicopathological features. Candidate miRNAs (miR-9, miR-433, miR-19b, and miR-370) were found strongly downregulated in GC tissues and cell lines. Their expression was increased following 5-AZA-CdR treatment. CpG island methylation of miR-9 was significantly higher in GC tissues compared to normal controls. After two demethylation treatments, miR-9 methylation degree was significantly decreased and miR-9 expression was ob-viously restored in GC cells and animal models. Deregulation of miR-9 was positively correlated with tumor lesion size. Three other miRNAs, miR-19b, miR-433, and miR-370 were assοciated with lymph node metastasis, decreased curvature, and poorly differentiated carcinoma. miR-19b and miR-433 were positively correlated with male gender. Of four candidate miRNAs downregulated in GC, miR-9 is epigenetically regulated by DNA methylation both in vitro and in vivo.
authors.affiliation: Chongqing Cancer Institute, Chongqing 400030, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Gastroenterology, Guizhou Provincial People's Hospital, The Affiliated People's Hospital of Guiyang Medical University, Guizhou 550002, P.R. China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Department of Surgery, The First People Hospital, Jiangbei, Chongqing 400020, P.R. China.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Gastroenterology, Children's Hospital, Chongqing Medical University, Chongqing 400014, P.R. China.
authors.forename: Zhengzheng
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, P.R. China.
authors.forename: Hongchun
authors.initials: H
authors.lastname: Luo
authors.affiliation: Laboratory of Clinical Diagnostics, Chongqing Medical University, Chongqing 400016, P.R. China.
authors.forename: Yuanhu
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Laboratory of Clinical Diagnostics, Chongqing Medical University, Chongqing 400016, P.R. China.
authors.forename: Zhizhong
authors.initials: Z
authors.lastname: Lu
authors.affiliation: Department of Gastroenterology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, P.R. China.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Wu
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D001374:Azacitidine / Q000008:administration & dosage / Q000031:analogs & derivatives; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D000077209:Decitabine; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008297:Male; D051379:Mice; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics*; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
chemical_list: C541874:MIRN92 microRNA, human; D035683:MicroRNAs; D000077209:Decitabine; D001374:Azacitidine