SiRNA稳定抑制肝癌细胞hTERT基因的表达

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与抑制肝癌细胞增殖的时-效关系．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并导入肝癌SMMC-7721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、MTF和PCR—TRAP法同时检测pGenesil—shRNA-hTERT稳定抑制组和未处理SMMC-7721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SMMC-7721细胞株中，RNAi效力持续、稳定存在，hTERTmRNA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 mRNA表达及肿瘤细胞增殖，是有潜力的基因治疗肿瘤新方法．

Stable inhibition of hTERT gene by siRNA in hepatocarcinoma cells

AIM: To elucidate the time-efficiency relation of stable inhibition of hepatocarcinoma cell 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 RNA (shRNA) targeting hTERT gene was designed, recombinant vector pGenesil-shRNA-hTERT was constructed and transfected into hepatocarcinoma SMMC-7721 cells, which were stably selected by G418 to establish hepatocarcinoma cell lines stablely expressing 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 hepatocarcinoma SMMC-7721 cells expressing stably pGenesil-shRNA-hTERT. In the cell lines expressing stably pGenesil-shRNA-hTERT, hTERT mRNA was obviously suppressed, and the telomerase activities were significantly decreased. Hepatocarcinoma SMMC-7721 cell proliferation significantly inhibited in pGenesil-shRNA-hTERT group compared to that in negative control group. CONCLUSION: RNAi may continually and stably suppress hTERT mRNA expression and carcinoma cell proliferation, which is a potential new approach for anti-tumor gene therapy.