RNAi双沉默Survivin和hTERT基因抑制人结肠癌SW480细胞增殖促进凋亡

摘要

Objective To investigate the Influence of Survivin and hTERT gene on cell proliferation and apoptosis in human colorectal carcinoma cell line SW 480 and to find experiment evidence for gene therapy of colorectal carci-noma .Methods Plasmids carrying shRNAs targeting survivin and hTERT were designed , constructed and trans-fected into SW480 cells.SW480 cells were then divided into blank group , blank Plasmid control group , survivin RNAi group , hTERT RNAi group and Survivin-hTERT RNAi group .The telomerase activity was examined by TRAP-PCR-ELISA analysis 48h after hTERT-shRNA transfection.Survivin and hTERT mRNA and protein expres-sion was analyzed by RT-PCR and Western blot .Cell apoptosis , proliferation were measured by flow cytometry , CCK-8 assay.Results Telomerase activity of SW480 cells in Survivin-hTERT RNAi groups were significantly decreased compared with the blank group ( P<0.01 ) .The expression of survivin and hTERT mRNA, proteins in the Survivin-hTERT RNAi group was reduced by 82.8%and 73.6%( P<0.01 ) ,79.2%and 66.7%( P<0.01 ) respectively .The inhibitory rate of cell proliferation of Survivin-hTERT RNAi group was 43.6% ±0.1%( P <0.01 ) .The apoptosis rate was 39.2%±2.3%( P<0.01 ) in the Survivin-hTERT RNAi group .Conclusions The Survivin-hTERT RNAi group could significantly reduces the protein expression of survivin and hTERT mRNA, in-hibit cell proliferation and induces cell apoptosis in human colorectal carcinoma cell line SW 480 .%目的：探讨双向沉默Survivin和hTERT基因对结肠癌SW480细胞增殖和凋亡的影响，为结肠癌的基因治疗提供实验依据。方法设计并构建能够稳定转录shRNA并可分别及联合干扰Survivin和hTERT分子表达的质粒，将其转染结肠癌SW480细胞后，分阴性对照组、空白质粒对照组、 Survivin RNAi 组、hTERT RNAi 组和Survivin-hTERT RNAi组。转染48h后TRAP-PCR-ELISA法检测端粒酶活性，RT-PCR法检测Survivin和hTERT mRNA的表达，Western blot检测Survivin和hTERT蛋白的表达，流式细胞仪及cck-8法分别检测细胞凋亡和细胞增殖的变化。结果 Survivin-hTERT RNAi组SW480细胞端粒酶活性明显下降（ P ＜0.01）， Survivin-hTERT RNAi 组Survivin及hTERT的mRNA水平较正常对照组分别减低82.8％和73.6％（ P ＜0.01），蛋白表达抑制率分别为79.2％和66.7％（ P＜0.01）。实验各组中Survivin-hTERT RNAi组细胞增殖抑制率和凋亡率最高，分别为43.6％±0.1％和39.2％±2.3％（ P＜0.01）。结论 Survivin和hTERT双干扰质粒可明显下调Survivin和hTERT蛋白的表达，抑制结肠癌SW480细胞的增殖，促进其凋亡。