Survivin靶向RNA干扰对人结肠癌HCT116细胞生长影响的研究

目的设计和构建survivin特异性的siRNA真核表达载体，观察survivin siRNA重组体对人大肠癌HCT116细胞株生长的影响，为大肠癌的基因治疗提供理论依据。方法针对survivin mRNA序列设计合成编码siRNA的DNA模板，构建2个survivin RNAi真核表达载体；实验分为survivin siRNA重组质粒转染组、空载体组和HCT116组，转染大肠癌细胞HCT116细胞，采用RT-PCR法检测survivin mRNA的表达，观察重组质粒对转染的HCT116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。结果测序表明survivin干扰序列完全正确，RT-PCR结果显示2条survivin siRNA真核表达质粒均能有效抑制survivin mRNA的转录表达。MTT比色法检测显示，与阴性对照组及未转染组细胞比较，干扰组细胞增殖率明显下降（P〈0．05）。流式细胞术检测的转染重组质粒组细胞凋亡率高于脂质体对照组和空质粒对照组（P〈0．05）。结论成功构建了survivin干扰真核表达载体，siRNA重组体能有效抑制人大肠癌细胞survivin mRNA表达，并抑制结肠癌细胞生长，促进其凋亡。

Inhibitory effect of silencing survivin gene with siRNA on growth of human colorectal carcinoma cell line HCT116

Objective To design and construct the RNAi eukaryotic vector of survivin gene and observe its interfering effect on the growth of human colorectal carcinoma cell line HCT116, and to provide evidence in gene treatment for Intestinal neoplasms. Methods DNA oligonucleotide for small interfering RNA expression targeting survivin gene was synthesized and inserted into pGenSil-1 , two recombinant plasmids （pRNAi-1, pRNAi-2） were constructed. The human colorectal carcinoma cell line HCT116 were divided into three groups： liposome treated control group, empty plasmid transfected control group and survivin RNAi transfected group. The expression of survivin mRNA was detected by semiquantitive RT-PCR. Methyl thiazolyl tetrazolium （MTT） assay was applied to determine the cell growth status. Apoptotie rates were evaluated by flow cytometry （FCM）. Results The sequence of specific siRNA was correct by sequence analysis. Compared with the negative control cells, RT-PCR showed the expression of surviving mRNA was down-regulated in the transfeeted cells （P 〈 0.05）. The MTT assay results showed that the subcloned recombinant plasmid expressing shRNA effectively inhibited HCT116 cell growth; the FCM results showed that the apoptotic rates in pRNAi-1, pRNAi- 2 transfected group were higher than those in liposome treated control group and empty plasmid transfected control group （P 〈 0.05）. Conclusion The siRNA eukaryotic expression vector of survivin gene have been constructed successfully which could effectively down-regulate the expression of survivin and proliferation in HCT116 cell line and induce apoptosis of colorectal carcinoma cells.