Methylation Regulating MiR-200b Expression And The Influence Of Gastric Cancer Cell Proliferation Invasive Ability

Objective: Gene abnormal methylation plays an important role in a variety of disease mechanisms. This research aims at studying the relation between methylation extent and expression quantity of miR-200 b,and the effects on proliferation,invasion,apoptosis of gastric cancer cell line MGC-803,BGC-823 in vivo.Methods: We chose normal gastric epithelial cell(GES-1) and gastric cancer cell(MGC-803,BGC-823) as research object, miR-200 b as the indicator. Extracting the total RNA from untreated GES-1,MGC-803 and BGC-823 to detect the changes of miR-200 b by real-time quantitative PCR, the promoter methylation of miR-200 b were detected by Bisulphite PCR. Then, MGC-803, BGC-823 cell line were treated with different concentration of 5’-Aza-CdR(2.5,5.0,7.5,10μmol/L) for 72 hours, set blank control group at the same time, the expression quantity and the promoter methylation of miR-200 b were detected by RT-PCR and Bisulphite PCR. Next, chose the appropriate concentration(10μmol/L) base on the results above and then for the next research. Finally, detect the changes of invasion ability, cell cycle and apoptosis of MGC-803,BGC-823 after treating with 10μmol/L 5’-Aza-CdR for 72 hours. The two results are tested by Transwell, flow cytometer respectively. The expression quantity of E-cadherin, N-cadherin, ZEB1, Slug, MMP9 are detected with Western blot. Test the above indicators again after treating with 10μmol/L 5’-Aza-CdR for 72 hours to analyze the potential role of miR-200 b in the process of epithelial mesenchymal transformation.Results: The expression quantity of miR-200 b in GES-1 was obviously higher than that in gastric cancer cells, The differences were statistically significant(p <0.05). The promoter methylation level of miR-200 b in GES-1 was lower than that in MGC-803 and BGC-823, The differences were statistically significant(p < 0.05).After treated with different concentration of 5’-Aza-CdR every 24 h for 72 h, the expression of miR-200 b of MGC-803 and BGC-823 cell lines were upregulation step by step. on the contrary, the DNA promoter methylation status were down-regulation at 10μmol/L. The differences were statistically significant(p < 0.05). When adding10μmol/L 5’-Aza-CdR in MGC-803, BGC-823 cells for 72 hours, the ability of invasion was decreased than that in untreated cell(p < 0.05), the cell cycle was arrested in G0/G1 phase(p < 0.05) and the apoptosis rate of two cancer cell lines were increased, the differences were statistically significant(p < 0.05). However,Western blot showed that the expression quantity of E-cadherin was increased when compared with that of untreated cell. But the expression quantity of N-cadherin,ZEB1, Slug, MMP9 were decreased when compared with that of untreated cell. The differences were statistically significant(p < 0.05). So the level of epithelial mesenchymal transformation was decreased when compared with that of control group.Conclution: The decreased expression of miR-200 b may play an important role in the process of gastric cancer. The mechanism may have some relationship with the methylation of miR-200 b promoter region. There are many methylation site during miR-200 b promoter region, miR-200 b is affected by different degrees of promoter methylation and impact gastric cancer cell line proliferation and invasion. Therefor,the relationship between epigenetics modification and the expression of miR-200 b will have potential therapeutic value in cancer therapy and profound significance for further research.