RNA干扰抑制肝癌QGY细胞株hTERT基因表达的研究

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.

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

Objective To elucidate the time-efficiency relation of stable inhibition of hepatoma cells proliferation by RNA interference 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 pGenesil-shRNA-hTERT.Real-time RT-PCR,MTT and PCR-TRAP were utilized to detect the alterations of hTERT mRNA expressions,telomerase activity and cell proliferation.Results The effectiveness of RNAi existed continually 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 pGenesil-shRNA-hTERT groups compared to negative control groups.Conclusion RNAi may continually and stably suppress hTERT mRNA expressions and neoplasm proliferation,which is a potential new approach for neoplasm gene therapy.