Research On The Retrovirus-mediated Antisense Human Telomerase RNA (hTR) Gene Induces The Apoptosis Of Hepatocellular Carcinoma

Objective:To investigate the inhibitory effect of antisense human telomerase RNA(hTR) gene on hepatocellular carcinoma in vitro,the long-term effect espically.Methods:The research team led by Professor Liu Jiyong preliminary got the recombinant retroviral vector plasmid PLXSN-hTR,carrying the retro-hTR gene.The procedure of plasmid extraction were routinely carried out,and the concentration and purity of the extracted plasmids were measured by UV spectrophotometer.Sense and antisense hTR gene were transfected into the packaging cell line PT67 by electroporation,and selected with G418 to get stable transfected cell lines.The recombinant retroviral supernantant were collected,filtered through 0.45μm filters, and concentrated 100-fold by centrifugation.The virus in the supernantant were titrated by transfecting NIH3T3 cell line.Then they were transfected into BEL-7402 cells.After G 418 selection,PCR was resorted to demonstrate the successful integration of the hTR gene.The sense and antisense hTR vectors were transfected into BEL-7402 cells respectively.Telemere repeat amplification protocol(TRAP-PCR-ELISA) was used to determine the Telomerase activity;cell cycle distribution and cell apoptosis were detected by the means of flow cytometric analysis. P53,Bax,Bcl-2 protein in transfected and untransfected cells was determined by Western blot。Results:The antisense hTR gene was shown to be successfully integrated into the target cells' genome,nergeticly;dye with Hochest 33258,compared with hTR sense clones and parental Bel-7402 cells,the fluorescence intensity of hTR antisense clones was weaker,and the positive rate was higher,Telomerase activity in the antisense hTR gene-treated group decreased significantly;FCM showed that the apoptosis rate of the experimental group increased,the proportion of Bel-7402 eels in G₂/M phase was higher and the proportion of cells in S phase was lower compared with the control groups,but no obvious diference of G₀/G₁ phase distribution was found.We detected the up-regulated expression of P53 and Bax protein,down-regulated expression levels of Bcl-2 protein.Conclusion:The antisense hTR can inhibit the telomerase activity of Bel-7402 and induce apoptosis.It can down-regulate the expression levels of telomerase as well as the expression levels of Bcl-2 protein, up-regulate theP53 and Bax protein,suggesting that antisense hTR gene can not only decrease the telomerase activity but also partially induce apoptosis in cancer cells.