肿瘤特异性Survivin、Livin共沉默RNAi载体的构建及其在前列腺癌细胞中的RNA干涉作用

The construction of recombinant RNAi vector of Survivin and Livin and inhibition Survivin and Livin gene expression in PC- 3 cells

目的:构建含Survivin基因启动子的、肿瘤特异性Survivin、Livin共沉默RNAi载体,研究该载体在前列腺癌细胞中的RNA沉默作用。方法:运用分子克隆技术,选取Survivin、Livin基因RNAi特异性序列,构建以Survivin、Livin基因为靶点的、含Survivin启动子的CGM30 miR-30 shRNA载体。经测序验证,用脂质体法转染PC-3细胞,RT-PCR、免疫印迹实验检测Survivin、Livin的表达变化,并用流式细胞术检测转染后PC-3细胞的凋亡变化。结果:构建的共沉默RNAi重组载体转染PC-3细胞后,Survivin、Livin基因在mRNA和蛋白水平上的表达均明显下调(P<0.01),且转染后的细胞凋亡增加了约15%,而正常前列腺上皮BPH-1细胞中则无此作用。结论:成功构建了含Survivin启动子、特异性沉默Survivin、Livin基因表达的RNAi共沉默载体CGM30-SP-svv-liv,转染该重组质粒后可促进肿瘤细胞的凋亡。

Objective：To construct the tumor specificity RNA interference vector which content the Survivin promoter to silence the expression of Survivin and Livin gene and promote the cell apoptosis in human prostate cancer cell PC-3.Methods：The CMV promoter of CGM30 miR-30 shRNA vector was replaced by Survivin promoter.Two target gene segments were synthesized and cloned into CGM30 miR-30 shRNA vector respectively,recombinant vectors were identified by enzyme digestion analysis and DNA sequencing.Then PC-3 cells were transfected with recombinant vectors and the interference effect was detected by RT-PCR and Western blot,the apoptosis index of PC-3 cells was detected by flow cytometry.Results：The results of RT-PCR and Western blot indicated that recombinant vectors could knock down the transcription and expression of Survivin and Livin gene only in PC-3 cells.After transfected with recombinant vectors,the apoptosis index of PC-3 cells was increased about 15％.Conclusion：The tumor specificity RNA interference recombinant vector for Survivin and Livin gene were constructed successfully,recombinant vector can promote the apoptosis of PC-3 cells effectively.