Study On Autophagy,Apoptosis And Mitochondrial Degradation In Nasopharyngeal Carcinoma Cells Induced By TIGAR

Objective: To investigate the effects occurred in autophagy,apoptosis,mitochondrial degradation and mitochondrial morphology and function of nasopharyngeal carcinoma 5-8F cells induced by knockdown of TP53-induced glycolysis and apoptosis regulator.Methods: 1 ? cultured nasopharyngeal carcinoma 5-8F cell line in vitro,the lenti-shRNA vector system against TIGAR was constructed,packed,and purified and then transfected 5-8F cell,and the expression of TIGAR in cytoplasm and mitochondria was detected by immunofluorescence analysis and Western blot;2?The contents of reduced glutathione(GSH),oxidized glutathione(GSSG)and reactive oxygen species(ROS)in cells after TIGAR interference were analyzed by flow cytometry to evaluate the levels of intracellular oxidative stress;3?The expression of autophagy-related proteins LC3,Beclin-1 and P62 were detected by Western blot to evaluate the autophagy activity;4?The cell was stained with JC-1 to analyze the mitochondrial membrane potential;5?The cytoplasmic protein and mitochondrial protein were respectively extracted to detect the change of cytochrome c expression in mitochondria and cytoplasm by Western blot.The apoptosis rate was detected by flow cytometry with Annexin V / PI staining cells;6?Western blot was employed to detect the expression of mitochondrial autophagy-related protein PINK1 and Parkin.Mitochondria were labeled with mitochondrial green fluorescent probe,the mass of mitochondria was analyzed by flow cytometry,and the expression of mitochondrial housekeeping protein-mtHSP70 was analyzed by Western blot to detect the Changes in the number of mitochondrial;7?The ultrastructure of the cells was observed under electron microscope,especially the morphological changes of mitochondria.The changes of intracellular ATP content were detected by high performance liquid chromatography(HPLC)to evaluate the mitochondrial function.Results: 1?The sh-TIGAR and sh-scramble cells were constructed successfully by lentivirus-shRNA transfection of 5-8F cells.Western blot and immunofluorescence analysis showed that The TIGAR expression was significantly decreased in cytoplasm and mitochondria of sh-TIGAR 5-8F cells;2?knockdown of TIGAR led to a decrease in intracellular NADPH production,reduced glutathione(GSH),and increased levels of ROS,eventually leading to the change of intracellular redox state;3?The expression of autophagy marker proteins LC3-II / LC3-I and Beclin1 was significantly increased in sh-TIGAR cells while the expression of P62 was decreased at the same time,which indicated that autophagy activity was enhanced;4?blocked TIGAR resulted in the decrease of mitochondrial membrane potential;5?The expression of cytochrome c in sh-TIGAR cells was significantly increased in cytoplasm while decreased in mitochondria,and the apoptosis rate of sh-TIGAR cells was significantly increased,indicating that the apoptotic activity increased;6?After knockdown of TIGAR,the level of PINK1 in cytoplasm was increased as the expression of Parkin was increased in mitochondria,indicating that the mitochondrial autophagy was activated through the PINK1/Parkin pathway.However,the mass of mitochondria in sh-TIGAR cells is more than that of sh-scramble cells;7?In sh-TIGAR cells,the mitochondria had an abnormal appearance including mitochondrial swelling,crista collapse and vacuolization.Furthermore,the ability to produce ATP was affected in these cells,indicating physiological dysfunction.Conclusion: Knockdown of TIGAR resulted in the decrease of intracellular NADPH production,reduced glutathione(GSH),and increased levels of ROS,eventually leading to the change of intracellular redox state,and enhance the autophagy activity,increase the apoptosis rate,decrease the mitochondrial membrane potential.The opening of the mitochondrial membrane permeability transmembrane pore activated the PINK1/Parkin-mediated mitochondrial autophagy pathway,However,the final degradation of mitochondria was hindered,resulted in intracellular accumulation of damaged mitochondria,which is characterized by structural disorders and physiological dysfunction.