Human Cancer Biology miR-200b and miR-200c as Prognostic Factors and Mediators of Gastric Cancer Cell Progression

Purpose:Thepurpose of this studywas to investigate the clinicopathologic significance andpotential role of miR-200b and miR-200c in the development and progression of gastric cancer. Experimental Design: We examined miR-200b and miR-200c expression in 36 paired normal and stomach tumor specimens, as well as gastric cancer cell lines, by quantitative real-time PCR. In addition, miR-200b and miR-200c were detected by ISH using gastric cancer tissue microarrays, and the association between miR-200b and miR-200c levels and clinicopathologic factors and prognosis were analyzed. A luciferase assay was conducted for target evaluation. The functional effects of miR-200b and miR-200c on gastric cancer cells were validated by a cell proliferation assay and cell invasion and migration assays. Results: miR-200b and miR-200c were downregulated in the gastric cancer specimens and cell lines tested. miR-200b and miR-200c levels were significantly correlated with the clinical stage, T stage, lymph node metastasis, and survival of patients. Ectopic expression of miR-200b and miR-200c impaired cell growth and invasion. In addition, when overexpressed, miR-200b and miR-200c commonly directly targeted DNMT3A, DNMT3B, and SP1 (a transactivator of the DNMT1 gene), which resulted in marked reduction of the expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B at the protein level. This effect, in turn, led to a decrease in global DNA methylation and reexpression of p16, RASS1A1, and E-cadherin via promoter DNA hypomethylation. Conclusion:Our findings suggest that miR-200b and miR-200c, as valuable markers of gastric cancer prognosis, may be a promising approach to human gastric cancer treatment. Clin Cancer Res; 19(20); 5602–12. 2013 AACR. Introduction An increasing number of studies have showed thatmicroRNAs (miRNA) can function as oncogenes or tumor suppressors and are often dysregulated in tumors (1, 2). In this regard, oncogenic miRNAs are frequently upregulated, whereas tumor suppressive miRNAs are frequently downregulated in tumors. The oncogenic miR-183/182/96 cluster of miRNAs is upregulated in a variety of tumors (3–5), and it regulates oxidative apoptosis and sensitizes gliomal cells to chemotherapy (6). In contrast, we previously reported that miR-34a is greatly downregulated in breast cancer cells and tissues and inhibits breast cancer proliferation and invasion (7, 8). In addition, miR-216b is greatly downregulated in nasopharyngeal carcinoma and inhibits tumor growth by targeting KRAS (9). The miR-200 family consists of 5 members (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) that are clustered and expressed as 2 separate polycistronic pri-miRNA transcripts with the miR-200b-200a-429 cluster at chromosomal location 1p36 andmiR-200c-141 cluster at chromosomal location 12p13. miR-200 is a miRNA family with tumor suppressive functions in a wide range of cancers, including breast cancer (10), colorectal cancer (11), pancreatic cancer (12), and endometrial carcinoma (13), but by now, the role of miR200 (miR-200a, miR-200b, andmiR-200c) in gastric cancer remained undefined. DNA methylation consists of an enzymatic addition of a methyl group at the carbon 5 position of cytosine in the context of the sequence 50-cytosine-guanosine (CpG) and is mediated by DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B; refs. 14 and 15). The promoter regions of approximately 50% of human genes contain regions of Authors' Affiliations: Department of Breast Oncology,Sun Yat-Sen University Cancer Center; State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center; Cancer Hospital and Cancer Research Institute, Guangzhou Medical University, Guangzhou, Guangdong; and Cancer Research Institute, University of South China, Hengyang, Hunan, China Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). H. Tang, M. Deng, and Y. Tang contributed equally to this work. Corresponding Authors: Xiaoming Xie, Department of Breast Oncology, State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, 651 East Dongfeng Road, Guangzhou 510060, China; Phone: 86-20-61639540; Fax: 86-20-38320368; E-mail: [email protected]; Qi Su, E-mail: [email protected] doi: 10.1158/1078-0432.CCR-13-1326 2013 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 19(20) October 15, 2013 5602 on July 15, 2017. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst August 30, 2013; DOI: 10.1158/1078-0432.CCR-13-1326