人端粒酶逆转录酶基因siRNA表达框架的构建和体外表达研究

Construction and selection of siRNA expression cassettes targeting human telomerase reverse transcriptase gene in vitro

目的探讨靶向小干扰RNA表达框架(SEC)对舌癌细胞内源性人端粒酶逆转录酶基因(hTERT)的特异性抑制作用。方法利用两步法PCR技术构建多条SEC,采用第5代聚酰胺-胺型树枝状高聚物(G5 PAMAM-D)纳米微球介导SEC转染Tca8113细胞,应用实时荧光定量RT-PCR技术和Western-blot等检测内源性hTERT的表达水平,并进一步筛选出最有效的小干扰RNA靶序列。结果针对不同靶位点设计的SEC干扰效果有显著差异,SEC-A的干扰效果最强,并且能以高度序列特异性方式沉默hTERT基因的表达;此外,转染次数与载体理化特性亦可影响SEC的干扰效应(P<0．05)。结论RNA干扰效应主要取决于靶序列;G5 PAMAM-D纳米载体能够介导SEC-A高效抑制内源性hTERT基因的表达;hTERT基因有望成为舌癌基因治疗的理想靶点。

Objective To determine whether the human telomerase reverse transcriptase （hTERT） gene silencing could be effectively induced by PCR-derived siRNA expression cassettes （SEC） transfected by the fifth generation polyamidoamine dendrimer （G5 PAMAM-D） in Tca8113 cells. Methods Four SEC were rationally designed and constructed based on a two-step PCR reaction. The SEC were then transferred into TcaS113 cells using G5 PAMAM-D, and hTERT expression was investigated by real-time fluorescence-quantitative reverse transcriptase-PCR and western blot analysis. Results The RNA interference effects of the SEC targeted for varying hTERT mRNA positions showed a significant disparity. Among them, SEC-A revealed the most potent inhibitory effects （above 95% of reduction）, followed by SEC-D and SEC-C, and SEC-B had no effect on hTERT expression （P 〉 0. 05 ）. That the endogenous hTERT gene silencing induced by G5 PAMAM dendrimer-mediated SEC-A was highly sequence-specific, and multiple transfection as well as properties of the vectors were routinely attributable to the specific suppression. Conclusions Specific inhibition of endogenous hTERT expression by use of a PCR-based short hairpin siRNA technique and dendrimer transfer system may serve as a novel strategy for treatment of tongue cancers expressing hTERT in vitro.