Epigenetic silencing of miRNA‑9 is correlated with promoter‑proximal CpG island hypermethylation in gastric cancer in vitro and in vivo

Li,Yan; Xu,Zhong; Li,Bo; Zhang,Zhengzheng; Luo,Hongchun; Wang,Yuanhu; Lu,Zhizhong; Wu,Xiaoling

doi:10.3892/ijo.2014.2667

Epigenetic silencing of miRNA‑9 is correlated with promoter‑proximal CpG island hypermethylation in gastric cancer in vitro and in vivo

Authors:
- Yan Li
- Zhong Xu
- Bo Li
- Zhengzheng Zhang
- Hongchun Luo
- Yuanhu Wang
- Zhizhong Lu
- Xiaoling Wu
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Affiliations: Chongqing Cancer Institute, Chongqing 400030, P.R. China, Department of Gastroenterology, Guizhou Provincial People's Hospital, The Affiliated People's Hospital of Guiyang Medical University, Guizhou 550002, P.R. China, Department of Surgery, The First People Hospital, Jiangbei, Chongqing 400020, P.R. China, Department of Gastroenterology, Children's Hospital, Chongqing Medical University, Chongqing 400014, P.R. China, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, P.R. China, Laboratory of Clinical Diagnostics, Chongqing Medical University, Chongqing 400016, P.R. China, Department of Gastroenterology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, P.R. China
Published online on: September 23, 2014 https://doi.org/10.3892/ijo.2014.2667
Pages: 2576-2586

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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 obviously 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.

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