A New Method To Prepare RNA Interference Vector And Its Inhibition To HTERT Of Tumor Cells In Vitro

Objective:To explore a novel vector for RNA interference on tumor hTERT gene expression, the U6/H1 double promoters siRNA/miRNA expression vectors were constructed by overlap extension PCR. The vectors were designed to target to three different regions of human telomerase hTERT gene exon and one was targeted to the untranslated region. The interfering efficacy on the gene expression was evaluated by telomerase activity in HepG2 cells and HeLa cells.Methods:four pairs of specific primer were designed based on the telomerase hTERT gene (genebank NM₁98253). The U6 and H1 promoter was amplified from pRNT-U6.1 plasmid and pSUPER by PCR respectively. And the siRNA/miRNA expression vectors were constructed by overlap extension PCR.After transfecting the purified U6/H1 double promters siRNA or miRNA expression vectors into HepG2 cells and HeLa cells with calcium phosphate co-precipitation procedures, the interefering efficacy of the expression vectors on the telomerase activity in the cells was assessed by TRAP-PCR-silver staining and gray scale scanning, and SYBRGreen quantitative fluorescence analysis.Results:2.5% agarose electrophoresis analysis of PCR product showed that single U6 or H1 promoter expression vectors was as the expected length of 366bp and 245bp respectively, and the U6/H1 double promters siRNA expression cassettes (SECs) was 592bp.The telomverase activity observed was significantly decreased after HepG2 cells and HeLa cells were transfected with the siRNA expression vectors (target to specific regions of hTERT gene,2650-2668, 2760-2778,3009-3027 and 3964-3982). Compared to the control, the telomerase activity inhibition rate was 75.00%,67.35%,68.46%,81.80% in HepG2 cells and 70.31%,36.80%,57.39%,80.47% in HeLa cells, respectively. Conclusion:The U6/H1 double promoters siRNA/miRNA expression vectors can be successfully constructed by overlap extension PCR, and the siRNA/miRNA expression vectors constructed were able to decreased the telomerase activity when transfected to hepG2 and HeLa cells in vitro. The prepareing method is fast and cost effective, and without any other unrelated exogenous sequences introduced. It can be applyed for screening effective siRNA/miRNA gene candidates and founding RNAi labraries. It also provides a new strategy for tumor therapy.