The PinX1 Gene Downregulates Telomerase And Induces Autophagy In Nasopharyngeal Carcinoma

PART ? the PinXl gene targeting telomerase induces autophagy in nasopharyngeal carcinomaObjective:To investigate autophagy and cell proliferation by the effect of PinXl targeting telomerase in nasopharyngeal carcinomaMethods:The expression of PinXl in CNE2 and 6-10B nasopharyngeal carcinoma cells was determined by qPCR and Western blot.Low-expressing cell lines were selected and transfected with PinX1 over-expressing plasmid and unloaded plasmid for experiment.RT-PCR was used to analyze hTERT mRNA expression in each group.Western blot and immunofluorescence were used to monitor autophagy expression.CCK-8 and transwell chambers were used to detect the cell proliferation,migration and invasion ability of each group.Flow cytometry was performed to examine changes in the cell cycle and apoptosis.Result:1.The expression of PinXl is lower in CNE2 cell line than in 6-10B cell line.2.After the over-expression plasmid was introduced into CNE2 cell line,the expression of PinX1 increased as well as the level of hTERT decreased.3.The expressions of LC3? and P62 increased in blank CNE2 group,as well as the level of LC3? and Beclinl decreased,and autophagy flux decreased.The proliferation,invasion and migration ability of cells were significantly increased.4.The expressions of LC3? and Beclinl in PinX1 over-expression group increased,as well as the level of LC3I and P62 decreased,and autophagy flux formation increased.The proliferation,invasion and migration ability of cells were significantly reduced in PinXl over-expression group.PinXl over-expression treatment also resulted in CNE2 cells cycle arrest at G0/G1 phase,and induced apoptosis.Conclusion:The expression of autophagy is inferior in CNE2 nasopharyngeal carcinoma cells.After PinXl targets telomerase,the expression of autophagy is enhanced.The proliferation,migration,and invasion capabilities of nasopharyngeal carcinoma cells are reduced,and apoptosis is increased.PART ? the molecular mechanism regulates autophagy in nasopharyngeal carcinoma cells by PinXl gene targeting telomeraseObjective:To investigate the molecular mechanism of autophagy by PinX1 gene targeting telomerase in nasopharyngeal carcinoma cellsMethods:The CNE2 cell line was transfected with the PinXl over-expression plasmid and the unloaded plasmid.Then an autophagy inhibitor 3-MA was added to the PinXl over-expression group.Western blot and immunofluorescence were used to monitor the expression of autophagy in each group.CCK8 and transwell cells were used to detect the cell proliferation,migration and invasion ability.Apoptosis was analyzed by flow cytometry.And then,western blot was used to anaylze the expression of p65/p-p65.Chloroquine was added to the PinXl over-expression group to analyze the expression of autophagy-related proteins p-AKT and p-mTOR.Result:1.When 3-MA was added to the PinXl over-expression group,the expression of LC3? and Beclinl decreased as well as the LC31 and P62 increased,and autophagy flux formation decreased.Cell proliferation,migration and invasion were enhanced,and apoptosis index decreased.2.The expression of p65/p-p65 increased in CNE2 cells,but decreased in PinXl over-expression group.When 3-MA was added into PinXl over-expression group,the expression of p65/p-p65 increased.3.In PinXl over-expression group,p-AKT and p-mTOR were significantly increased.After addition of chloroquine,the autophagy-related proteins p-AKT and p-mTOR were not significantly inhibited,but the formation of autophagy flux is reduced.Conclusion:The PinXl gene may induce autophagy through AKT/mTOR pathway in nasopharyngeal carcinoma cells.The activation of autophagy promotes apoptosis in the same time,highly possible by blocking NF-?B/p65.PART ? the Pinxl gene targeting telomerase inhibits the growth of tumor spheres in xenografts miceObjective:To investigate the effection of PinXl in the proliferation of nasopharyngeal carcinoma by nude mice experiment Methods:The CNE2 celsl,CNE2 cells transfected with empty plasmid and CNE2 cells transfected with PinX1 over-expression plasmid were inoculated into three groups of nude mice,and draw the tumor growth curves in each group.The nuclear morphology in each group was observed by HE staining.The expression of PinXl in each group was detected by immunohistochemistry.Result:1.The tumors of nude mice in PinXl over-expression group were significantly inhibited compared with the other two groups.2.The nucleus of HE sections in each group showed different degrees of atypia.Immunohistochemistry showed that the expression of PinXl in PinX1 over-expression group was significantly higher than that of other two groups.Conclusion:PinXl gene significantly inhibited the growth of nasopharyngeal carcinoma cells in nude mice,supporting its potential as a therapeutic target for nasopharyngeal carcinoma.