RNAi技术沉默Bmi-1基因对人胃癌AGS细胞株的作用

Role of RNAi of Bmi-1 Gene in Gastric Carcinoma AGS Cells

目的:探讨RNA干扰(RNAi)技术沉默Bmi-1基因表达对人胃癌细胞株AGS增殖和侵袭能力的影响。方法:利用慢病毒表达体系pHelper1.0/pHelper2.0/pGCL-GFP,构建针对Bmi-1基因shRNA序列的RNAi重组质粒vshRNA-Bmi-1,转染AGS细胞,采用Western blot检测重组质粒对Bmi-1蛋白表达的影响,MTT法检测重组质粒对AGS细胞体外生长的抑制作用,PI单染法流式细胞术检测转染重组质粒后细胞凋亡与细胞周期的变化,小室侵袭实验检测AGS细胞侵袭能力变化。结果:成功构建了vshRNA-Bmi-1重组质粒,并成功转染AGS细胞抑制Bmi-1蛋白的表达。转染重组质粒后,AGS细胞增殖明显受到抑制,细胞凋亡增加,S期比例上升,细胞侵袭能力弱于非特异性转染组。结论:vshRNA-Bmi-1重组质粒明显下调Bmi-1蛋白在AGS胃癌细胞中的表达,并抑制肿瘤细胞的增殖与侵袭,促进其凋亡。

Objective：To investigate the effects of RNA interference （RNAi） to Bmi-1 on the proliferation,apoptosis,and invasion ability of human gastric carcinoma cell line AGS. Methods：Recombinant plasmid vshRNA-Bmi-1 was transfected into AGS cells,and the cells untransfected and those transfected with non-specific shRNA were used as control. After AGS cells were transfected for 72 h,the expression of Bmi-1 mRNA and protein was detected by real time quantitative PCR （RT-PCR） and Western blot. MTT and flow cytometry were used to observe the cell proliferation and apoptosis. The invasion ability was evaluated by cell invasion assay kit. Results：vshRNA-Bmi-1 was constructed successfully,expression of Bmi-1 mRNA and protein in AGS cells with specific transfection was weaker than in those with non-specific transfection and in those without transfection （all P0.05）; invasive ability were inhibited in AGS after specific transfection（P0.01）; down-regulation of Bmi-1 expression significantly induced the apoptosis of AGS cells （P0.05）. Conclusion：RNAi-mediated down-regulation of Bmi-1 gene inhibits the invasion and growth of human gastric carcinoma AGS cells,and induces the apoptosis of AGS cells. It may provide a novel way for the treatment of gastric carcinoma