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目的探讨沉默高尔基体α-甘露糖苷酶Ⅱ(Golgiα-mannosidaseⅡ,GMⅡ)基因的表达对人胃癌BGC-823细胞增殖与凋亡的影响。方法根据GenBank中登录的人GMⅡ基因mRNA序列及siRNA设计原则,设计了4条siRNA,并构建4个针对靶点的重组表达质粒pGPU6/GFP/Neo-1140、pGPU6/GFP/Neo-1303、pGPU6/GFP/Neo-1406和pGPU6/GFP/Neo-1817,同时合成阴性对照质粒pGPU6/GFP/Neo-shNC,经Lipofectamine 2000分别转染BGC-823细胞,通过RT-PCR及Western blot检测转染细胞中GMⅡ基因mRNA和蛋白的表达,筛选沉默效果最佳的质粒;经MTT试验、细胞周期分布分析、Hoechst33258和Annexin V-FITC/PI双染试验分别检测沉默GMⅡ基因对人BGC-823细胞增殖与凋亡的影响。结果 pGPU6/GFP/Neo-1303组GMⅡ基因mRNA和蛋白的表达明显低于空白对照组和阴性对照组(P<0.05),表明pGPU6/GFP/Neo-1303组沉默效果最好;与空白对照组和阴性对照组相比,pGPU6/GFP/Neo-1303组BGC-823细胞增殖抑制率明显升高(P<0.01),G1期细胞比例明显上升(P<0.05),S期细胞比例明显下降(P<0.01),细胞凋亡率明显升高(P<0.05)。结论已成功沉默BGC-823细胞中GMⅡ基因的表达,从而抑制BGC-823细胞的增殖并促进其凋亡,GMⅡ可能成为防治胃癌的新靶点。
Objective To investigate the effects of silencing Golgiα-mannosidase Ⅱ (GMⅡ) gene expression on proliferation and apoptosis of human gastric cancer cell line BGC-823. Methods Four siRNAs were designed according to the human GMⅡ gene mRNA sequence and siRNA design principle registered in GenBank. Four recombinant plasmids pGPU6 / GFP / Neo-1140, pGPU6 / GFP / Neo-1303 and pGPU6 The negative control plasmid pGPU6 / GFP / Neo-shNC was synthesized and transfected into BGC-823 cells by Lipofectamine 2000. The transfected cells were detected by RT-PCR and Western blot The expression of GMⅡgene mRNA and protein was detected by RT-PCR. The plasmid with the best silencing effect was screened by MTT assay, cell cycle distribution analysis, Hoechst33258 and Annexin V-FITC / PI double staining. Effect of apoptosis. Results The expression of GMⅡ mRNA and protein in pGPU6 / GFP / Neo-1303 group was significantly lower than that in blank control group and negative control group (P <0.05), which indicated that pGPU6 / GFP / Neo-1303 group had the best silencing effect. Compared with blank control group Compared with the negative control group, the proliferation inhibition rate of BGC-823 cells in pGPU6 / GFP / Neo-1303 group was significantly increased (P <0.01), the proportion of cells in G1 phase was significantly increased (P <0.05) P <0.01), the apoptosis rate was significantly increased (P <0.05). Conclusion The expression of GMⅡ gene in BGC-823 cells has been silenced, and the proliferation and apoptosis of BGC-823 cells have been inhibited. GMⅡ may be a new target for the prevention and treatment of gastric cancer.