The Relationship And Possible Mechanism Between Autophagy And The Radio Sensitivity Of Nasopharyngeal Carcinoma

Part I The effect of ATG5 silencing on radiosensitivity of nasopharyngeal carcinoma CNE-1 and CNE-2 cells[Objective]To investigate the function of autophagy in radiosensitivity of NPC cells,specific lentivirus-delivered shRNA to ATG5 was used to induce ATG5 silencing,and the effect of ATG5 silencing on IR-induced cell viability,apoptosisg and DNA damage were detected.[Methods]Specific lentivirus-delivered shRNA to ATG5 was used to induce ATG5 silencing in CNE-1 and CNE-2 cells.ATG5 mRNA expression was detected by real-time PCR while the ATG5 protein level was determined by western blot analysis to confirm the effect of ATG5 silencing.To investigate the effect of ATG5 silencing on radiosensitivity of CNE-1 and CNE-2 cells,cell viability was measured by the CCK-8 assay,percentage of apoptotic cells was assessed by flow cytometry and the protein level of ?-H2AX(?-histone family 2A variant,y-H2AX)to confirm the DNA damage was evaluated by western blot analysis.[Results]Real-time PCR analysis and western blot analysis revealed that silencing of ATG5 markedly decreased the expression of ATG5 mRNA(P<0.01)and protein in CNE cells compared with the non-targeted shRNA cells and control cells.CCK8 assay showed that after silencing of ATG5,cell viability of CNE cells significantly decreased compared with that of the scrambled and control group(P<0.05).Results of flow cytometry showed that CNE cells transfected with the ATG5-targeted shRNA had a higher proportion of apoptotic cells compared to that of cells transfected with the non-targeted shRNA cells and control cells(P<0.05).Western blot analysis showed that the protein level of y-H2AX in the CNE cells was increased in all experimental groups after IR.However,a higher increase in the protein level of ?-H2AX was observed in ATG5 silencing groups.[Conclusion]Specific lentivirus-delivered shRNA to ATG5 effectively inhibits the expression of ATG5 and inhibition of autophagy enhances the radiosensitivity of CNE cells.Part II Study on the mechanism of autophagy inhibition in regulating the radiosensitivity of nasopharyngeal carcinoma CNE-1 and CNE-2 cells[Objective]In this part,we investigated the influence of autophagy inhibition on cell cycle distribution and the mRNA expression of RAD51,analyzed the effect of RAD51 on cell viability and elucidated the possible mechanism of autophagy in regulation of radiosensitivity.[Methods]Cell cycle distribution was analyzed by flow cytometry and RAD51 mRNA expression was detected by real-time PCR,while the cell viability was measured by the CCK-8 assay.The cells of autophagy inhibitor treated groups were treated with 3-MA or CQ 2 h before irradiation.The cells of RAD51 inhibitor treated groups were treated with B02 or RI-1 1 h before irradiation.The groups of RAD51 overexpression were transfected by flag-pcDNA3-RAD51 plasmid withLipofectamineTM 2000 48 h before irradiation.[Results]Cell cycle analysis by flow cytometric measurement revealed that no difference in cell cycle distribution was noted in all experimental groups in CNE-1 cells.However,suppression of ATG5 led to cell cycle arrest in the G1 phase while the percentage of cells in the G2/M phase was decreased dramatically in CNE-2 cells compared with that of the scrambled and control group(P<0.05).Real-time PCR analysis revealed that the expression of RAD51 mRNA demonstrated a notable rise in the negative control and control cells when treated with radiation(P<0.01).However,when cells were transfected with the ATG5-targeted shRNA,the RAD51 mRNA expression revealed a significant decline both in cells treated with or without radiation compared with that of the negative control and mock-transfected control cells(P<0.05).Moreover,a greater decrease in the expression of RAD51 mRNA was observed as the cells were subjected to a combined treatment with radiation(P<0.01).The influence of autophagy inhibition on the expression of RAD51was ascertained by treating cells with 3-MA and CQ(P<0.01).After irradiation,a marked decrease in cell viability was observed both in cells transfected with the ATG5-targeted shRNA alone or scrambled cells treated with RAD51 inhibitor compared to the cell viability of the scrambled cells(P<0.01).However,no change was noted between the cells transfected with the ATG5-targeted shRNA and the non-targeted shRNA when cells were pre-treated with B02 or RI-1.The viability of the cells transfected with the ATG5-targeted shRNA was significantly decreased compared to that of the mock-transfected control cells,but showed an increase when cells were transfected with the flag-RAD51 vector before irradiation(P<0.01),which reversed the enhanced radiosensitivity by ATG5 suppression in CNE cells.[Conclusion]Suppression of autophagy does not accelerated cell cycle progression in human nasopharyngeal carcinoma cells but enhances the radiosensitivity of nasopharyngeal carcinoma cells by reducing RAD51 expression,which plays an important role in homologous recombination in resynthesizing the damaged region of the DNA.