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

RNAi-silenced Survivin and h TERT gene inhibits proliferation and induces apoptosis of human colorectal carcinoma cell line SW480

目的探讨双向沉默Survivin和h TERT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h TERT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、h TERT RNAi组和Survivinh TERT RNAi组。转染48h后TRAP-PCR-ELISA法检测端粒酶活性,RT-PCR法检测Survivin和h TERT mRNA的表达,Western blot检测Survivin和h TERT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-h TERT RNAi组SW480细胞端粒酶活性明显下降(P<0.01),Survivin-h TERT RNAi组Survivin及h TERT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-h TERT 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h TERT双干扰质粒可明显下调Survivin和h TERT蛋白的表达,抑制结肠癌SW480细胞的增殖,促进其凋亡。

Objective To investigate the Influence of Survivin and h TERT gene on cell proliferation and apoptosis in human colorectal carcinoma cell line SW480 and to find experiment evidence for gene therapy of colorectal carcinoma. Methods Plasmids carrying shRNAs targeting survivin and h TERT were designed,constructed and transfected into SW480 cells. SW480 cells were then divided into blank group,blank Plasmid control group,survivin RNAi group,h TERT RNAi group and Survivin-h TERT RNAi group. The telomerase activity was examined by TRAP-PCR-ELISA analysis 48 h after h TERT-shRNA transfection. Survivin and h TERT mRNA and protein expression 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-h TERT RNAi groups were significantlydecreased compared with the blank group( P < 0. 01). The expression of survivin and h TERT mRNA,proteins in the Survivin-h TERT 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-h TERT RNAi group was 43. 6% ± 0. 1%( P <0. 01). The apoptosis rate was 39. 2% ± 2. 3%( P < 0. 01) in the Survivin-h TERT RNAi group. Conclusions The Survivin-h TERT RNAi group could significantly reduces the protein expression of survivin and h TERT mRNA,inhibit cell proliferation and induces cell apoptosis in human colorectal carcinoma cell line SW480.