体外构建双H1启动子SECs及其对HepG2细胞端粒酶活性的干扰作用

The interfering effect of construction of double-H1 promoters SECs on telomerase gene expression in HepG2 cells

目的构建针对端粒酶hTERT的双H1启动子SECs,并探讨其转录生成的siRNA对HepG2细胞端粒酶活性的抑制作用。方法利用融合PCR技术,构建2个针对人端粒酶hTERT基因外显子不同片断的双H1启动子SECs,分别转染人肝癌细胞HepG2,转录生成siRNAs。用TRAP法和PCR-EIA法检测端粒酶活性,分析双H1启动子SECs对HepG2细胞端粒酶表达的干扰作用。结果成功构建针对hTERT的双H1启动子SECs,其转录产物siRNA可明显抑制HepG2细胞端粒酶的活性。结论siRNA SECs对HepG2细胞端粒酶hTERT基因表达有明显的干扰作用,为临床开展对肿瘤端粒酶基因干扰抑制的实验研究奠定了基础。

Objective To construct the double-H1promoters siRNA expression cassettes （SECs） targeted to human telomere retrotranscriptase （ hTERT ） , and investigate the interfering effect of its siRNA on hTERT gene expression in HepG2 cells. Methods SECs were constructed by fusing PCR, based on two different human telomerase hTERT gene fragments. When SEGs transferred into HepG2 cells respectively, the SECs were transcripted to the siRNA. The interfering effect of SECs on the telomerase activity in the cells was assessed by telomeric repeat amplification protocol （TRAP） and PCR-EIA. Results SEGs were successful constracted, and the telomerase activity was significantly inhibited when the HepG2 cells were tranfected with SECs. Conclusions The siRNA SECs display a definite RNA interference effect on the expression of telomerase. This method of SECs preparation can be applied for RNAi research in tumor inhibition.