RNA干涉下调survivin基因对人肝癌细胞的生长抑制及促凋亡作用

Effect of down-regulating survivin expression by RNAi on growth suppression and promotion on apoptosis of human hepatocellular carcinoma cells

目的:探讨RNA干涉技术下调survivin基因表达对人肝癌细胞的生长抑制及促凋亡的作用,为肝癌的基因治疗提供有效靶点及技术。方法:构建pSU6-si-survivin重组质粒,转染人肝癌细胞株Bel7402,设空白组、阴性组和治疗组,采用MTT法检测肝癌细胞的增殖活性,RT-PCR检测survivin基因mRNA水平,Western blotting及免疫组化法检测survivin蛋白表达,流式细胞技术(FCM)及AO/EB染色观察细胞凋亡。结果:MTT法检测,治疗组24、48和72 h细胞生长抑制率(%)分别为21.43±1.67、39.07±3.80和59.72±7.05,与阴性组比较差异有显著性(P<0.01)。RT-PCR及Western blotting检测,治疗组细胞survivin基因在mRNA及蛋白水平上表达均下降,上述产物量与β-actin的比值与空白组和阴性组比较差异均有显著性(P<0.01)。FCM检测治疗组细胞阻滞于G0/G1期,凋亡率为(20.65±1.28)%,与空白组及阴性组比较差异有显著性(P<0.01)。AO/EB双染色治疗组细胞呈早中期凋亡。结论:重组质粒pSU6-si-survivin可抑制survivin基因在人肝癌细胞中的表达,抑制肝癌细胞生长,促进肝癌细胞凋亡;survivin基因可作为肝癌基因治疗的重要靶点。

Objective To explore the effect of down-regulating survivin expression by RNAi on growth suppression and promotion on apoptosis of human hepatocellular carcinoma cells, in order to supply an effective target and technique for liver cancer gene therapy. Methods The recombinant plasmid of pSU6-si-survivin was constructed, which was transfected into Be17402 cells. Mock group, negative group and treated group were set up. Using MTT assay the proliferation of transfected cells was investigated, the survivin protein expression level was determined by Western blotting and immunohistochemical staining. Transcription of survivin was detected by semi-quantitative RT-PCR. Flow cytometry （FCM） and AO/EB double dyeing were used to examine apoptosis in transfected ceils. Results MTT assay demonstrated that the cell growth inhibition rates （%） of transfected cells in treatedgroup at 24, 48 and 72 h were 21.43±1.67, 39.07±3.80, and 59.72±7.05, there were significant differences compared with negative group （P〈0. 01）. The results of RT-PCR and Western blotting showed that the mRNA and protein levels of survivin declined markedly in transfected cells, the ratio of expression product to β-actin in treated group had significant differences compared with negative group and mock group （P 〈 0.01）. The results of FCM indicated that the cells in treated group were inhibited in G0/G1 stage, the apoptotie rate was 20.65%±1.28%, there were significant differences compared with mock group and negative group （P〈0.01）. AO/EB double dyeing showed that the most cells in treated group were in early or middle stage apoptosis. Conclusion The recombinant plasmid of pSU6-si-survivin can significantly inhibit the expression of survivin gene in Be17402 cells, and suppress cell growth and induce apoptosis, so survivin may act as an important target to treat human hepatocellular carcinoma.