hTERT-siRNA表达载体的构建及对MCF-7细胞生长、端粒酶活性的抑制作用

Construction of hTERT-siRNA expression vector and its inhibitory effects on the growth of MCF-7 cells and the activity of telomerase

目的构建人端粒酶逆转录酶(hTERT)基因的RNA干扰(RNAi)表达载体,并研究该载体对乳腺癌MCF-7细胞端粒酶活性及细胞增殖的影响,为针对端粒酶的乳腺癌基因治疗提供新的途径。方法设计针对人端粒酶逆转录酶催化亚基(hTERT)的干扰靶序列TGTTCAGCGTGCTCAACTA,构建重组siRNA表达质粒pGenesil-hTERT,同时构建不针对任何基因的阴性对照重组pGenesil-HK。两种重组质粒经酶切、电泳分析和测序鉴定后,用脂质体转染法分别转染乳腺癌MCF-7细胞,应用端粒酶重复序列扩增聚合酶链反应(TRAP-PCR)及聚丙烯酰胺凝胶电泳检测端粒酶活性,流式细胞仪测定细胞凋亡率。结果酶切电泳测序分析表明插入序列正确,重组质粒构建成功。转染pGenesil-hTERT的MCF-7细胞,凝胶电泳见端粒酶特征性条带明显减少,端粒酶活性受到明显抑制;pGenesil-hTERT转染细胞后凋亡率较对照组明显升高(P<0.01),且转染48h凋亡率最高,达54.7%±2.41%。结论hTERT-siRNA可有效抑制乳腺癌MCF-7细胞端粒酶活性、促进细胞凋亡,此法有望应用于肿瘤基因治疗。

Objective To construct the RNAi expression vector of hTERT gene, and to investigate the inhibitory effects of the vector on the growth of mammary adenocarcinoma cell MCF-7 and the activity of telomerase. So as to present a new approach to the gene therapy for breast cancer which aim at inhibiting the telomerase activity. Methods An interference target sequence TGTTCAGCGTGCTCA ACTA which aimed at hTERT gene was designed and the siRNA expression plasmid pGenesil-hTERT was constructed by the means of recombination. Meanwhile a negative control recombinant-pGenesil-HK that did not aim at any gene was constructed by the same method. The recombinants expression vectors pGenesil-hTERT and pGenesil-HK were identified by electrophoresis after digestion and DNA sequencing. The two recombinants were transfected into the MCF-7 cells by lipofectamineTM 2000. The telomerase activity was tested by telomerase repeat sequence amplification and polyacrylamide gel electrophoresis. Flow cytometry was used to detect the cell apoptosis. Results The results of analysis and the sequencing identification confirmed that the target sequence was inserted into the predicted site precisely and the recombining plasmids were successfully constructed. The PAGE results showed that the mark traps of telomerase were obviously decreased and the telomerase activity of pGenesil-hTERT was obviously inhibited. The apoptotic rate of the pGenesil-hTERT group was significantly higher than that of the pGenesil-HK group （P〈0. 01）, the highest apoptosis rate was （54. 7±2. 41） % at the 48th hour. Conclusion hTERT-siRNA can inhibit the expression of human telomerase and proliferation of MCF-7 cells. It may open a new approach to the use of RNAi as a new tool to study gene function in cancer cell lines, and may be developed to be a new gene therapeutic agent for cancer.