Livin和Survivin基因短发夹RNA真核表达载体的构建及其联合转染对肝癌细胞增殖和凋亡的影响

Construction of eukaryotic expression vector of short hairpin RNA of Livin and Survivin gene and its effect of combined transfection on the proliferation and apoptosis of hepatoma cells

目的设计并构建能够有效干扰Livin和Survivin基因的表达,且能在胞内稳定转录的表达载体;研究Livin shRNA和Survivin shRNA的联合转染对肝癌细胞增殖活性及凋亡的影响。方法根据设计siRNA的原理,分别从Livin、Survivin基因序列中选取两段siRNA靶序列模板,以其为基础进行shRNA真核表达载体Pgenesil-1-Livin和Pgenesil-1-Survivin的构建。将传代培养的肝癌Hep G2细胞株分为五组:设阴性对照组(不转染)、空白质粒组(单独转染Pgenesil-1-NC)、Survivin组(单独转染Pgenesil-1-Survivin)、Livin组(单独转染Pgenesil-1-Livin)和共转染组(联合转染Pgenesil-1-Livin和Pgenesil-1-Survivin),对各组细胞进行单独或联合转染。为检测转染2 d后Livin和Survivin蛋白表达的情况、转染后三个不同时间点(48 h、60 h、72 h)的细胞增殖活性变化及转染后48 h的细胞凋亡率,分别采用了蛋白质印迹法、MTT法及TUNEL法。结果两种表达载体的测序结果与预期相同。与阴性对照组及空白质粒组相比,转染后2 d,Livin组、Survivin组和共转染组的蛋白表达均明显下降(P<0.05),而与Livin组和Survivin组相比,共转染组蛋白表达的下降更为显著(P<0.05);各组细胞在3个检测时间均表现出增殖活性的下降,且与Livin组和Survivin组相比,共转染组在三个检测时间的细胞生长抑制率(IR)值分别为28.541±0.842、21.644±0.129、15.532±1.12,明显高于前者(P<0.05);转染后48 h,共转染组凋亡率为53.956%±4.332%,明显大于Livin组和Survivin组(P<0.05)。结论成功设计并构建体内可稳定转录且能有效干扰目的基因表达的质粒载体。对肝癌细胞进行Livin shRNA和Survivin shRNA的联合转染,可更大程度下调相应基因的表达,降低Livin蛋白和Survivin蛋白的胞内含量,可更为有效地阻碍癌细胞的异常增殖,促进癌细胞的程序性凋亡。

Objective To design and construct plasmids capable of interfering with Livin and Survivin gene expression and capable of stable transcription of short hairpin RNAs and to study the effect of combined transfection on the proliferation and apoptosis of HCC cells. Methods According to the principle of designing siRNA,two siRNA target sequences were selected from Livin and Survivin gene sequences. Construction of shRNA eukaryotic expression vectors Pgenesil-1-Livin and Pgenesil-1-Survivin was based on these two sequences. HepG2 cells were subcultured in five groups（ negative control group,blank plasmid group,Survivin group,Livin group and co-transfection group）. Each group was transfected alone or co-transfected. In order to analyze the expression of Livin and Survivin protein after transfection for 2 days,the cell proliferation activity at three time points after transfection and the apoptotic rate after transfection for 48 h were determined by Western blotting,MTT and TUNEL respectively. Results The sequencing results of the two expression vectors were the same as expected. Compared with untransfected and transfected only blank plasmids,the protein expression of the separate transfected group and the combined transfection group decreased significantly,while compared with Livin group and Survivin group,the decrease of anti-apoptotic protein expression was more significant. The cells showed a decrease in proliferation activity at three detection time（ 48 h,60 h,72 h）. Compared with Livin group and Survivin group,the IR of the three transfection groups were 28. 541 ± 0. 842,21. 644 ± 0. 129 and 15. 532 ± 1. 12,respectively,significantly higher than the former（ P〈0. 05）.The apoptotic rate of co-transfection group was 53. 956% ± 4. 332%,significantly greater than the Livin group and Survivin group. Conclusion Successful designed and constructed plasmid vectors that can be stably transcribed in vivo and capable of interfering with the expression of the gene of interest and transfection of Livin shRNA and Survivin shRNA in Hepatocellular Carcinoma Cells can inhibit the intracellular expression of the two target genes to a greater extent,reduce the intracellular content of Livin protein and Survivin protein,hinder the abnormal proliferation of cancer cells more effectively and promote the apoptosis of cancer cells more significantly.