Effect Of SiRNA Targeting The Subunit Of Telomerase On Hepatocellular Carcinoma

Liver cancer is a familiar malignant tumor. However, there is no good therapeutic strategy yet. The mostly often used strategy today is the combination of several therapies including chemotherapy and gene therapy. By using the method of TRAP, Kim et al reported that high telomerase activity could be detected in liver cancer cells, while there was no telomerase activity in normal liver cells. Therefore, it seems that the telomerase could be a target for the diagnosis and therapy of the liver cancer.Telomeres, the DNA-protein complexes at the ends of eukaryotic chromosomes, are protective against genome instability-promoting events. The lenghth of the telomere relates to the telomerase activity .The telomerase of human contains hTERT, hTR and hTEP-1. Among them, hTERT and hTR is necessary for the function of the telomerase.In this article, RNA interference was used to down-regulate the gene expression of hTR and hTERT. The effect of the siRNA was studied. The telomerase activity, the proliferation and apoptosis of the hepatoma cell after the down-regulation of the subunit of telomeras were also examined. Our work on controlling the telomerase activity by RNAi in human hepatoma cell provided a possible strategy for the design of new anti liver cancer drugs.We designed and chemically sythesized three siRNA targeting the hTERT gene, and synthesized the DNA templates for another two siRNA by PCR and then subcloned them into pEGFP vector. We also designed three siRNA targeting the hTR gene and subcloned their DNA templatesinto pEGFP vector as above. Then the synthetic siRNA or the siRNA expressing vectors were transfected into the Bel-7404 liver cancer cells by oligofectamin or lipofectamin 2000 respectively. The efficiency of transfection was examined with fluorescent microscope. The cells with stable siRNA expression were picked out by using G418. Then the totle RNA of the cells was extracted by using Trizol reagent, and the expression level was examined by RT-PCR. The telomerase activity was detected by TRAP. The apoptosis was examined by FACS. The expression level of PEG10 and hTERT was examined by real time quantitative PCR.All of the eight siRNA could downregulate the genes expression level with different efficiency. The siRNA expressed by the vectors had the most effective inhibition after 24 hours, while the chemically synthesized siRNA was most effective after 48 hours. The telomerase activity was atenuated when the genes expression was down-regulated. Obvious apoptosis of the cells was observed 7 days after transfection, while little apoptosis was observed in the unspecific siRNA treated cells and the untreated cells. We obtained the stable transfected cells which showed little telomerase activity. The expression of hTERT was up or down-regulated according to the expression of PEG10.