Survivin-pPRIME-IGF1R-miR30慢病毒载体的构建及其对肝癌细胞生长的抑制

Construction of survivin-pPRIME-IGF1R-miR30 lentiviral vector and its inhibitory effect on proliferation of liver cancer cells

目的:探讨survivin启动子调控的重组慢病毒survivin-pPRIME-IGF1R-miR30载体(简写为sur-IGF1R-miR30)对肝癌Hep3B细胞IGF1R表达和细胞生长的影响。方法:PCR扩增survivin启动子,构建sur-pPRIME;将针对IGF1R基因的干扰序列与sur-pPRIME载体连接,构建sur-IGF1R-miR30慢病毒载体。将sur-IGF1R-miR30、psPAX2和pMD2G质粒共转染293T细胞,扩增慢病毒,检测病毒滴度。以sur-IGF1R-miR30感染人肝癌Hep3B细胞和胎肝L-02细胞,RT-PCR、Western blotting检测Hep3B细胞IGF1R的表达,CCK-8法检测Hep3B细胞的生长。结果:成功构建survivin启动子调控的慢病毒载体sur-IGF1R-miR30,滴度为4.58×109PFU/ml。Sur-IGF1R-miR30感染后,Hep3B细胞中特异表达荧光蛋白,L-02细胞中基本没有表达。Sur-IGF1R-miR30感染Hep3B细胞可阻断IGF1RmRNA和IGF1R蛋白的表达。Sur-IGF1R-miR30感染抑制肝癌细胞的生长,第7天时的抑制率达60%(P<0.05)。结论:成功构建的重组慢病毒载体sur-IGF1R-miR30可有效地降低肝癌细胞中IGF1R的表达,抑制肝癌细胞的增殖。

Objective To study the effects of survivin-promoter-regulated survivin-IGF1R-miR30（sur-IGFIR-miR30） lentiviral vector on the IGF1R expression and proliferation of hepatoma Hep3B cells.Methods：Survivin promoter was amplified by PCR and sur-pPRIME plasmid was constructed.Interference sequence targeting IGF1R gene was synthesized and cloned into sur-pPRIME plasmid,named sur-IGF1R-miR30.Sur-IGF1R-miR30,psPAX2,and pMD2G were co-transfected into 293T cells to amplify lentivirus,and the lentivirus titer was examined.IGF1R expression in Hep3B cells was detected by RT-PCR and Western blotting analysis,and the proliferation of Hep3B cells was evaluated by CCK-8 assay.Results：Sur-IGF1R-miR30 lentiviral vector regulated by survivin promoter were successfully constructed,and the virus titer was 4.58×109 PFU/ml.Fluorescent protein after sur-IGF1R-miR30 infection was expressed in Hep3B cells,but not in L-02 cells.Sur-IGF1R-miR30 infection inhibited IGF1R mRNA and protein expressions in Hep3B cells and the proliferation of Hep3B cells,with the inhibitory rate being 60% at 7 d（P〈0.05）.Conclusion： Sur-IGF1R-miR30 lentiviral vector can inhibit IGF1R expression and hepatoma cell proliferation.