RNA干扰抑制肝癌QGY细胞株hTERT基因表达的研究  

Inhibition of hTERT gene by RNA interference in hepatoma cell line QGY

作　　者：蒋明东[1] 李少林[2] 鄢勇[1] 王正洪[1] 方亮[1] 黄小波[1] 赵渝[1] 

机构地区：[1]重庆市第九人民医院肿瘤科,400700 [2]重庆医科大学核医学教研室,400016

出　　处：《重庆医学》2007年第20期2052-2054,F0004,共4页Chongqing medicine

摘　　要：目的利用RNA干扰稳定筛选-抑制技术,抑制人端粒酶逆转录酶(hTERT)基因表达,探讨靶向hTERT基因RNAi对肝癌细胞增殖的抑制效应。方法设计靶向hTERT基因的小干扰RNA,构建重组表达质粒pGenesil-shRNA-hTERT并导入人肝癌细胞系QGY细胞株,经G418筛选,建立稳定表达siRNA-hTERT的细胞株。采用real time RT-PCR、MTT和PCR-TRAP法同时检测pGenesil-shRNA-hTERT稳定抑制组和未处理QGY细胞组hTERT基因表达、端粒酶活性及细胞增殖变化。结果在稳定表达pGenesil-shRNA-hTERT的QGY细胞株中,RNAi效力持续、稳定存在,hTERT的mRNA表达、端粒酶活性明显降低,瘤细胞增殖被抑制。结论RNA干扰能持续、稳定地抑制靶基因hTERT的mRNA表达及肿瘤细胞增殖,是潜在的肿瘤基因治疗新方法。Objective To elucidate the time efficiency relation of stable inhibition of hepatoma cells proliferation by RNA inter ferenee in vitro. Methods The stable screening-inhibition technique of RNAi was adopted to suppress the expression of hTERT stably. After small hairpin interfering RNA （shRNA） targeting hTERT gene was designed,recombinant vector pGenesil shRNA hTERT were constructed.and transfected into hepatoma QGY cells which were stably selected by G418 to establish hepatoma cell lines of stable expression pGencsil-shRNA-hTERT, Real time RT PCR,MTT and PCR-TRAP were utilized to detect the ahera tions of hTERT mRNA expressions,telomerase activity and cell proliferation, Results The effectiveness of RNAi existed continu ally and stably in hepatoma QGY cells expressing stably pGenesil shRNA-hTERT. In the cell lines expressing stably pGenesil shRNA hTERT,hTERT mRNA was obviously suppressed.the telomerase activities were significantly decreased. Hepatoma QGY cell proliferation significantly inhibited in pGenesii shRNA hTERT groups compared to negative conlroi groups. Conclusion RNAi may continually and stably suppress hTERT mRNA expressions and neoplasm proliferation,which is a potential new approach for neoplasm gene therapy.

关 键 词：RNAI 肝癌细胞株 HTERT 稳定抑制 质粒 

分 类 号：R735.7[医药卫生—肿瘤] R730.21[医药卫生—临床医学]