Specific Expression Of SiRNA-telomerase Driven By Htert Promoter Inhibits The Growth Of HeLa Cells

Background and AimsRNA interference is the sequence-specific gene-silencing induced by double-strand RNA (dsRNA), and gives information about gene function quickly, easily, and inexpensively. RNAi can block the expression of target gene in transcript level, post-transcript level and protein level. As a power gene silencing technology, high effectively and high specific is the most important character of RNAi. Though the use of RNAi for genetic-based therapies is widely studied, especially in viral infections, cancers, and inherited genetic disorders. More and more studies still suggested that non-specific effects can be induced by siRNAs, such as off-target inhibition, activation of interferon response, and saturation of cellular silencing machinery. This will badly affect the usage of RNAi in gene therapy. It has been known that more than90%of human tumors exhibit telomerase activity. Consequently, telomerase is believed to be a broad-spectrum molecular marker of malignancies. In the present study we attempt to develop a tumor-specific RNAi system using the human telomerase reverse transcriptase promoter. It was expected to limit the expression of siRNA in tumor cells, not in normal cells. This system may provide a basis for RNAi therapy. Next we constructed the recombine plasmid which can express the siRNA targeting the telomerase, and then studied the effects of siRNA-telomerase on the growth of Human cervical carcinoma cell HeLa.MethodsThe plasmid pShuttleA-htertp-siRNA-GFP was constructed and transfeced into HeLa cells, and the expression of siRNA was detected with RT-PCR.then we cotransfected the plasmid pShuttleA-htertp-siRNA-GFP with pCDNA3.1/EGFP, which can express the green fluorescent protein into tumor cells HeLa and HEK-293human embryonic kidney cell line, and the normal human fibroblasts BJ, to detective the change of the expression level of fluorescent protein. The plasmid pShuttleA-htertp-siRNA-telomerase was also constructed and transfected intoHeLa cells, we detectived the effection of siRNA-telomerase on the growth of HeLa cells with soft-colony formation and plate-colony formation. ResultsWith RT-PCR we can detectived the expression of siRNA-GFP in the HeLa cells transfected with pShuttleA-htertp-siRNA-GFP, about70bp,but in the cells transfected woth control plasmid,there is no siRNA was detected. Subsequently, we observed that in HeLa cells and293cells transfected with pShuttleA-htertp-siRNA-GFP the expression of fluorescent protein (GFP) were decreased, and the inhibition rate was69%and75%, respectively.But in human normal fibrosis cells BJ, the expression of GFP has no change. With soft-agarose colony formation and plate colony formation, we identified that the growth of HeLa cells were inhibited after transtfected with pShuttleA-htertp-siRNA-telomerase, and the rate was42.9%.ConclusionsOur results indicate that hTERT promoter can induce the specific expression of siRNA in tomor cells, and has the effective interference, but not in the normal human fibroblasts BJ. After transfection the plasmid pShuttleA-htertp-siRNA-telomerase which expresses the siRNA targeting telomerase, the growth of HeLa cells was inhibited.