The Study On Htert Independent Of Telomerase In Regulating The Apoptosis Of Laryngeal Cancer Cells

Objective:To explore the mechanism of telomerase-independent anti-apoptosis of hTERT, investigated the relationship between the expression of human telomerase reverse transcriptase (hTERT) and telomerase activity or prostate apoptosis response4(Par-4) proteins in laryngocarcinoma cells after applying RNA interference to silencing the expression of hTERT gene in laryngeal carcinoma cells in vitro and in vivo, which supply an new method for the laryngocarcinoma gene therapy.Methods:A plasmid termed pEGFP-shTERT was constructed. Hep-2cells were transfected with the plasmids. After0,12,24,48hours, cells were collected and detected with TRAP-PCR in telomerase activity, with flow cytometer in cell apoptosis, with and immunocytochemistry in expression of hTERT and Par-4protein and with Western-blot in expression of hTERT and Par-4protein; An animal model of laryngocarcinoma was constructed. The plasmids pEGFP-shTERT were transfected by multipoint injection. The expression of Par-4and hTERT after transfected of0,6,10,14days was detected by Quantum dots-based immunofluoresense technology in xenograft laryngocarcinoma from nude mice.Results:The plasmids were successfully transfected after12h of treated with pEGFP-shTERT, the transfection rate achieved78.6%after24h. After24h of plasmids transfection, the telomerase activity of Hep-2cells started to decline but not be inhibited completely, while the apoptosis of Hep-2cells occurred before the inactivation of the telomerase after12h of plasmids transfection. The expression of hTERT was down-regulated and the expression of Par-4was increased after RNAi, and there are obvious dose-effect relationship (P<0.05); Meanwhile, the similar results were found in vivo study, furthermore before hTERT gene silencing, the hTERT protein were found both in nucleus and cytoplasm, while Par-4protein only expressed in cytoplasm. After14d of RNAi, the expression of hTERT protein decreased both in nucleus and cytoplasm, especially in nucleus, while the expression of Par-4protein obviously increased in nucleus.There was negative correlation between Par-4and hTERT expression in nucleus (P<0.05, r=-0.908).Conclusion:After24h of plasmids transfection, the telomerase activity of Hep-2cells started to decline but not obviously, while the apoptosis of Hep-2cells occurred before the inactivation of the telomerase. After24h of plasmids transfection, there were16.5% Hep-2cells in apoptosis. So, we consider that hTERT may have anti-apoptosis effect independent of telomerase. There was negative correlation between Par-4and hTERT expression when silencing hTERT gene, which meant Par-4may took part in the hTERT mediated apoptosis independent of telomerase. hTERT may interact with Par-4in cytoplasm and inhibit the translocation of Par-4into nucleus, and then inhibit the Par-4induced apoptosis.