α-甘露糖苷酶Ⅱ基因沉默对人胃癌BGC-823细胞增殖及凋亡的影响

Effect of Golgi α-mannosidaseⅡ gene silence on proliferation and apoptosis of human gastric carcinoma BGC-823 cells

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 effect of silencing Golgi α-mannosidase Ⅱ（GMⅡ）gene expression on the proliferation and apoptosis of human gastric carcinoma BGC-823 cells.Methods Four siRNA sequences were designed according to the human GMⅡ gene mRNA sequence in GenBank and the principle of siRNA design,based on which four plas mids expressing short hairpin RNA（shRNA）for silencing GMⅡ gene,i.e.pGPU6 / GFP / Neo-1140,pGPU6 / GFP / Neo-1303,pGPU6 / GFP / Neo-1406 and pGPU6 / GFP / Neo-1817,as well as negative control plasmid pGPU6 / GFP / Neo-shNC,were constructed and transfected to BGC-823 cells in mediation of lipofectamine 2000.The expressions of GMⅡ mRNA and protein in transfected BGC-823 cells were determined by RT-PCR and Western blot respectively,based on which the plasmid with optimal silencing effect was screened.The effect of GMⅡ gene silence on proliferation of BGC-823 cells was evaluated by MTT test and analysis of cell cycle distribution,while that on apoptosis of the cells by Hoechst 33258 and Annexin V-FITC / PI staining.Results The expression levels of GMⅡ mRNA and protein in BGC-823 cells transfected with plasmid pGPU6 / GFP / Neo-1303 were significantly lower than those in blank and negative control groups,indicating the optimal silencing effect of the plasmid.Compared with those in blank and negative control groups,the inhibiting rate of proliferation of BGC-823 cells transfected with the plasmid increased significantly（P 〈 0.01）,while the percentage of cells at G1 phase increased significantly（P 〈 0.05）and that at S phase decreased significantly（P 〈 0.01）.and the apoptosis rate of cells（P 〈 0.05）.Conclusion The expression of GMⅡ gene in BGC-823 cells were silenced successfully,while the proliferation of BGC-823 cells were inhibited and their apoptosis was promoted,suggesting that GM Ⅱ might be a novel target for treatment of human gastric carcinoma.