siRNA沉默survivin对人胃癌SGC-7901细胞生长的抑制作用

Inhibitory effect of silencing survivin gene with siRNA on growth of human gastric cancer SGC-7901 cells

目的:探讨siRNA沉默survivin基因表达对胃癌细胞生长的抑制作用及其机制,为胃癌及survivin阳性肿瘤的治疗提供理论依据。方法:针对survivin mRNA序列设计合成编码siRNA的DNA模板,构建2个重组质粒pGCsilencerU6/GFP/survivin siRNA-1和-2;实验分为脂质体对照、空质粒转染对照及survivin siRNA重组质粒转染治疗组,转染胃癌细胞SGC-7901,采用RT-PCR法检测survivin mRNA的表达,Western blotting检测蛋白表达,观察重组质粒对其转染的SCG-7901细胞survivin基因表达的影响;用四甲基偶氮唑盐(MTT)法测量细胞生长情况;用流式细胞术检测细胞凋亡情况。结果:Western blotting、RT-PCR结果证实转染重组质粒pGCsilenerU6/GFP/survivin-siRNA-1的SCG-7901细胞survivin蛋白质及mRNA抑制率(78.25%及88.75%)均高于脂质体对照组(5%及2%)和空质粒对照组(1%及6%)(P<0.01);转染重组质粒pGCsilenerU6/GFP/survivin-siRNA-2的SCG-7901细胞survivin蛋白质及mRNA抑制率(42%及77%)也均高于脂质体对照组和空质粒对照组(P<0.01);MTT法检测结果显示转染重组质粒pGCsilenerU6/GFP/survivin-siRNA-1组细胞体外生长的抑制率(64%)高于空质粒对照组(11%)(P<0.01);流式细胞术检测的转染重组质粒pGCsilenerU6/GFP/survivin-siRNA-1组细胞凋亡率(21.20±3.21)高于脂质体对照组(0.830±0.001)和空质粒对照组(1.98±0.67)(P<0.01)。结论:重组质粒pGCsilencerU6/GFP/survivin-siRNA可抑制胃癌细胞中survivin的表达,并抑制胃癌细胞生长,促进其凋亡。

Objective To investigate the inhibitory effect of silencing survivin gene with siRNA on the growth of gastric cancer cells and its mechanism, and provide evidence in treatment for gastric cancer. Methods DNA template coding survivin-specific siRNA was designed and synthesized. Two recombinant plasmids （pGCsilencerU6/GFP/survivin-siRNA-1 and -2） were constructed. The gastric carcinoma cell line SGC-7901 were divided into three groups： liposome-treated control group, empty plasmid-transfected control group and survivin-siRNA-1 transfected group. In order to observe the effect of survivin-siRNA, the expressions of survivin mRNA and protein were detected by RT-PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium （MTT） assay was applied to determine the cell growth status. Apoptotic rates were evaluated by flow cytometry （FCM）. Results The results of Western blotting and RT-PCR indicated that the inhibitory rates of protein and mRNA in pGCsilenerU6/GFP/survivin-siRNA-1 transfected grouo （78.25% and 88.75%） were higher than those in liposome-treated control group （5%and 2%） and empty plasmid-transfected control group （1% and 6%） （P〈0.01）. The inhibitory rates of protein and mRNA in pGCsilenerU6/GFP/survivin-siRNA-2 transfected group （42% and 77%） were higher than those in liposome-treated control group and empty plasmid-transfected control group also （P〈0.01）. The MTT assay results showed that the inhibitory rate of cell proliferation in survivin-siRNA-1 transfected group （64%） was higher than that in empty plasmid-transfected control group （11%） （P〈0.01） ; The FCM results showed that the apoptotic rate in pGCsilenerU6/GFP/survivin-siRNA-1 transfected group （21.20±3.21） was higher than those in liposome-treated control group （0. 830±0. 001） and empty plasmid-transfected control group （1.98±0.67） （P〈0.01）. Conclusion pGCsilencerU6/GFP/survivin-siRNA can not only suppress the expression of Survin in gastric cancer ceils and proliferation of SGC-7901 cells, hut also induce apoptosis of gastric cancer cells.