Inhibition HTERT By SiRNA Expression Cassettes With U6/H1 Promoters In HepG2 In Vitro

Objective:Construct siRNA expression cassettes(SECs)with an convergent opposing U6/H1 double promoter by the gene splicing overlap extension PCR based on three different human telomerase hTERT gene mRNA fragments,and evaluate the interfering efficacy of the convergent opposing U6/H1 double promoter SECs on the telomerase expression in HepG2 cells.Methods:The H1 and U6 promoter was amplified from pSUPER and pRNT-U6.1 plasmid by PCR,respectively.The specific primers were designed based on the telomerase hTERT gene(genebank NM₁98253).And siRNA expression cassettes(SECs)were constructed by overlap extension PCR.After transfecting the purified PCR products into HepG2 cells with calcium phosphate co-precipitation procedures, the hTERT mRNA was quantified by real-time fluorescence RT-PCR. The interfering effect of SECs on the telomerase activity in the cells was assessed by TRAP-silver staining.Results:The length of the first stage PCR products(single-U6 promoter sense/antisense SECs)and the third step PCR products(U6/H1 double promoter SECs)were confirmed as expecting(335bp,245bp of the former,respectively and 561bp of the latter)by agarose gel electrophoresis analysis.After the SECs were transfected into the HepG2,the hTERT mRNA expressin was significiengtly decreased,with the inhibitory rate 37.0%,50.1%å’Œ37.0%,respectively.The telomerse activity was also effectively inhibited,the relative telomerase activity declined by 55.0%,76.5%and 47.6%.While there was no significant remarkable change observed in negative control group in telomerase activity and hTERT mRNA expression level.Conclusion:The siRNA expression cassettes based on the telomerase hTERT gene with convergent opposing U6/H1 double promoter can be successfully constructed by gene splicing by overlap extension PCR,which may become a potential tool for hTERT gene expression silencing in tumors.The preparing method is fast and cost efffective,without any other unrelated exogenous gene introduced,and it could be used as an alternative method for screening effective siRNA gene candidates and screening siRNA gene libraries.