Methylation of miR-200b and its effects on proliferation,invasion of gastric cancer cell line MGC-803 / 天津医药

ABSTRACT

Objective To investigate the effect of expression level and methylation level of miR-200b on proliferation, invasion and apoptosis of gastric cancer cell line MGC-803 in vitro. Methods Normal human gastric epithelium cell line GES-1, and gastric cancer cell line MGC-803 cells were cultured and harvested to extract total RNA. Then miR-200b ex?pression level was examined via q-PCR;Methylation in promoter of miR-200b was revealed by Bisulphite PCR. MGC-803 cells were treated with different concentrations of 5′-Aza-CdR to test its effect on miR-200b expression and methylation of its promotor. The effect of its treatment at 10μmol/L for 72 h on invasion,proliferation and apoptosis of both cell lines were detected by Transwell assay, Flow cytometry and apoptosis assay respectively. The gene related to EMT (epithelial-mesen?chymal transition ) such as E-cadherin,N-cadherin,ZEB1,Slug were checked by Western blot. Results The expression of miR-200b in GES-1 is higher than that in MGC-803(P=0.022). The methylation level in promoter region of miR-200b in GES-1 is lower than that in MGC-803(P=0.034). After treated with 5′-Aza-CdR, the expression of miR-200b in MGC-803 cell was up-regulated in a timely dependent manner. On the contrary, the methylation in promoter of mirR-200b were down-regulated when 5′-Aza-CdR were added at 10μmol/L(P=0.043). What’s more, 5′-Aza-CdR administration increas?es cell apoptosis especially in aged cells and delays cell cycle at G0/G1 which in turn decrease ratio of cells in S phages. 5′-Aza-CdR treatment also decreased invasiveness and induced expression of E-cadherin, as well as down regulated the ex?pressions of N-cadhrin, ZEB1, Slug and MMP9 in MGC-803 cells. Conclusion Expression of miR-200b could be affected by methylation of promoter of miR-200b and in turn regulate proliferation and invasion of gastric cells in vitro.