The Role And Mechanism Of DNA Damage Repair Related Protein Mediated Alveolar Epithelial EMT In Radiation-induced Pulmonary Fibrosis

Objective: By means of experiments to explore the role of DNA damage repair related protein DNA-PKcs in radiation-induced EMT.To further elucidate the role and mechanism of DNA damage repair protein DNA-PKcs mediated by radiation induced EMT.So as to provide the basis for the biological function of DNA-PKcs in radiation-induced pulmonary fibrosis.Methods: Using Western blot,immunofluorescence and flow cytometry at different doses or at different time to detect the epithelial markers and mesenchymal markers and to build the irradiation induced epithelial mesenchymal transition model by60 Co γ-ray irradiation of A549 cells.Western blot,immunofluorescence and flow cytometry were used to detect the effect of knockdown of DNA-PKcs protein on radiation-induced EMT,and to explore whether DNA-PKcs mediated the occurrence of EMT induced by ionizing radiation.Western blot assay was used to detect the changes of the upstream transcription factor of EMT induced by knocking down protein of DNA-PKcs,and to speculate the mechanism of DNA-PKcs mediated radiation induced EMT.The use of Western blot detection in DNA-PKcs knockdown cell line can reverse the effect of DNA-PKcs knockdown on radiation-induced EMT to determine whether DNA-PKcs affects EMT through twist by knocking down twist.And the assay was used to detect the changes of twist expression and the degradation rate of twist protein after DNA-PKcs knockdown,and to explore the regulation of DNA-PKcs on the stability of twist.The interaction between DNA-PKcs and twist was detected by immunoprecipitation assay.Thus,it is speculated that DNA-PKcs possibly mediates the mechanism of EMT induced by irradiation.Results: 1.The results of Western blot showed that60 Co γ-ray irradiation of A549 cells,with the increase of irradiation time and irradiation dose,epithelial markers were reduced gradually,mesenchymal marker gradually increased at 6Gy,48 h was the most obvious,the results of immunofluorescence and flow cytometry results show that the radiation can induce lung epithelial cells EMT.2.The results of Western blot,immunofluorescence and flow cytometry showed that after knocking down DNA-PKcs,compared with the control group,the radiation induced EMT was further promoted.3.The results of Western blot showed that knockdown of twist in DNA-PKcs knockdown cell line was able to reverse the EMT induced by knocking down DNA-PKcs,which indicated that DNA-PKcs could affect EMT by twist.4.The results of co-immunoprecipitation showed that the interaction between DNA-PKcs and twist.5.Western blot results showed that after DNA-PKcs being knocked down,the expression of twist protein was up-regulated and the degradation rate slowed down,indicating that DNA-PKcs could regulate the stability of twist.Conclusions: 1.Ionizing radiation can induce lung epithelial cells to produce EMT.2DNA-PKcs mediated the occurrence of radiation-induced EMT.3.DNA-PKcs may regulate the degradation of EMT by regulating the degradation of twist by ubiquitination.