Construction Of Dual Promoters SECs In Vitro And Its Interfering Effect On Telomerase Activity In HepG2Cells

Objective:To construct the H1/U6dual promoters SECs targeting to human telomerase reverse transcriptase (hTERT), investigate the interfering effect of the siRNA/miRNA on hTERT gene expression in HepG2cells and access their efficacy.To explore a new way for making interfering RNA,and provide a new method for the treatment of human tumors.Methods:Using fusion PCR,two H1/U6dual promoters SECs targeting open reading frame of hTERT gene (1800～1818and2650～2668) and the other two of the SECs targeting the3’untranslated region of hTERT gene (3896～3982and3964～3982) were constructed.A SEC with randomized interference DNA sequence was constructed as the controlled.After transferred HepG2cells respectively by TurboFect transfection reagent or calcium phosphate co-precipitation protocol,the SECs were transcripted to the siRNA or miRNA.The telomerase activity was detected by TRAP-PCR-sliver and gray scale scanning to calculate the inhibition rate.The interfering effect of SECs on the telomerase activity in the cells was also assessed by Real-time TRAP-PCR directly.Results:The results of2.5%agarose electrophoresis and sequencing demonstrated that the SECs were successfully constructed.SECs targeting to the sites1800～1818,2650～2668,3896～3982and3964～3982were transferred to HepG2cells respectively with TurboFect transfection reagent.Compared to the control,the inhibition ratio of telomerase activity was42.38%,61.80%,77.95%,79.51%respectively and with calcium phosphate co-precipitation protocol the ratio was20.19%,27.38%,42.53%,64.3%.Conclusions:SECs can be constructed successfully and have obvious interfering effect on hTERT gene expression after transfecting HepG2cells.This approach may provide a new method for the synthesis of interfering RNA and explore a new idea to high-throughput screen effective interference sites and build RNAi libraries.It may also lay a foundation for clinical research on interfering tumors telomerase gene. There are12figures,10tables and50references.