人胃癌细胞BGC823中miR-200c靶基因产物波形蛋白的检测及功能研究

Functional study and detection of vimentin produced by miR-200c target gene in human gastric cancer BGC823 cells

目的:采用RNA靶向干扰技术,研究人胃癌细胞BGC823中微小RNA(microRNA,miRNA,miR)-200c的靶点锌指E盒增强子结合蛋白2(zinc finger E-box enhancer binding protein 2,ZEB2)基因的表达情况,并检测相关的波形蛋白基因表达情况,观察miR-200c对胃癌BGC823细胞功能的影响。方法:构建ZEB2基因表达的干扰质粒shZEB2,用Lipofectamine 2000转染胃癌BGC823细胞,用G418筛选稳定转染细胞株,进行实时定量聚合酶链反应(polymerase chain reaction,PCR)检测人BCG823细胞中ZEB2 mRNA、miR-200c及波形蛋白基因的表达水平,并用蛋白印迹法检测ZEB2蛋白、波形蛋白表达水平,噻唑蓝(methylthiazolyldiphenyl-tetrazolium bromide,MTT)法检测细胞增殖能力;Transwell小室检测细胞侵袭能力,划痕实验检测细胞迁移能力。结果:干扰ZEB2分子表达后,miR-200c表达水平增高,而BGC823细胞的增殖、迁移及侵袭能力明显下降。结论:ZEB2是miR-200c的检测靶点,干扰ZEB2后,胃癌细胞BGC823的增殖、迁移及侵袭能力明显降低。

Objective:To investigate the expression of zinc finger E-box enhancer binding protein 2(ZEB2)gene in miR-200c in human gastric cancer BGC823 cells by RNA targeted interference technique,and detect expression of vimentin,so as to observe the effect of miR-200 c on the function of gastric cancer BGC823 cells.Methods:The interference plasmids shZEB2 was constructed and transfected to BGC-823 cells in mediation of Lipofectamine 2000.The stable transfectants were screened by G418,and ZEB2 mRNA,miR-200 c and vimentin was detected by real-time PCR in BCG823 cells,while protein expression levels of ZEB2 and vimentin by Western blotting.Proliferation of BGC-823 cells was evaluated by MTT assay.Transwell assay and wound healing assay were used to observe the invasion and migration ability of BCG823 cells,respec-tively.Results:With interfering ZEB2 expression,miR-200c expression level increased while proliferation,migration and invasion ability of BGC823 cells decreased significantly.Conclusions:ZEB2 may be the target of miR-200c,and the ability of BGC823 cells including proliferation,migration and invasion is significantly decreased after interference with ZEB2.