摘要
AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.
AIM: To investigate the effects of antisense humantelomerase RNA (hTR) on the biologic behavior of humangastric cancer cell line: MKN-45 by gene transfection and itspotential role in the gene therapy of gastric cancer.METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryoticexpression vector (pEF6/V5-His-TOPO) in cis-direction ortrans-direction by DNA recombinant methods. Theconstructed sense, antisense and empty vectors weretransfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drugselection, the expression of antisense hTR gene in stabletransfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptoticfeatures by PI and Hoechst 33258 staining, the cell cycledistribution by flow cytometry and the population doublingtime by cell counting. Comparison among the stabletransfectants and normal MKN-45 cells was made.RESULTS: The sense, antisense hTR eukaryotic expressionvectors and empty vector were successfully constructed andproved to be the same as original design by restrictionendonuclease analysis and sequencing. Then, they weresuccessfully transfected into MKN-45 cell lines separatelywith lipofectin. The expression of antisense hTR gene wasonly detected in MKN-45 cells stably transfected withantisense hTR vector (named as MKN-45-ahTR) but not inthe control cells. In MKN-45-ahTR, the telomerase activitywas inhibited by 75 %, the apoptotic rate was increased to25.3 %, the percentage of cells in the G0/G1 phase wasincreased to 65 %, the proliferation index was decreased to35 % and the population doubling time was prolonged to 35.3hours. However, the telomerase activity, the apoptotic rate,the distribution of cell cycle, the proliferation index and thepopulation doubling time were not different among the controlcells.CONCLUSION: Antisense hTR can significantly inhibittelomerase activity and proliferation of MKN-45 cells andinduce cell apoptosis. Antisense gene therapy based onteiomerase inhibition can be a potential therapeuticapproach to the treatment of gastric cancer.
基金
the National Natural Science Foundation of China,No.39770725
