The Role And Mechanism Of DNA-PKcs In Radiation-induced Pulmonary Fibrosis And Screening And Verification Of Potential Therapeutic Targets For Radiation-induced Pulmonary Fibrosis

Objective:The role and molecular mechanism of DNA-PKcs in the development of radiation-induced pulmonary fibrosis（RIPF）were investigated by ⁶⁰Coγ-ray whole chest irradiation in DNA-PKcs knockout mice.At the same time,the potential therapeutic targets of RIPF were screened and verified by chemical drug intervention or gene therapy in RIPF mice,which provided important theoretical basis for the protection and treatment of RIPF.Methods:（1）DNA-PKcs knockout mice(DPK^-/-)were constructed by Cas9/sg RNA technology and subjected to 20 Gy whole chest irradiation.One month later,the differentially expressed genes were screened by mRNA sequencing of lung tissues,and the bioinformatics was used for functional analysis.（2）Lung tissue was collected one month and five months after irradiation,and the changes of inflammatory factors in lung tissue were detected by qPCR.H&E and Masson staining were used to compare the effect of DNA-PKcs deletion on the pathological changes of lung tissue induced by radiation.（3）The expressions of epithelial mesenchymal transformation（EMT）-related proteins in lung tissue,such as SPC,a-SMA,E-cadherin,N-cadherin and Twist,were detected by immunofluorescence and Western blot.（4）DNA-PKcs stable knockdown lung epithelial cell line（A549 shDPK）was constructed by lentivirus system,and the effect of DNA-PKcs deletion on Twist half-life was detected by actinomycin.The point mutation plasmid was constructed to explore the interaction between DNA-PKcs and Twist by immunoprecipitation.（5）In vitro experiments explored the role of AKT1 activity in the regulation of Twist by DNA-PKcs,and verified the correlation between AKT1 activity and Twist expression in lung tissue.（6）Intervention of DNA-PKcs targeting drug VND3207 by gavage before whole chest irradiation in mice.H&E and Masson staining were used to observe the effect of VND3207 on radiation-induced lung tissue pathological changes.（7）The phosphorylation activity of DNA-PKcs and the formation of g-H2AX focus in lung tissue and/or A549 cells were detected by immunofluorescence.Cloning formation test,EDU⁺test and apoptosis detection were used to detect whether VND3207alleviates radiation-induced cell damage by targeting DNA-PKcs.（8）To detect whether VND3207 alleviates radiation-induced EMT by targeting DNA-PKcs/AKT1/Twist.（9）To verify the role and mechanism of miR-486-3p in radiation-induced EMT in vitro.（10）The potential role of miR-486-3p carried by adeno-associated virus（AAV）in the prevention and treatment of RIPF.Results:（1）Lung tissue mRNA sequencing results showed that the differentially expressed proteins caused by DNA-PK deficiency were involved in cell metabolism,stress response and cell adhesion function,and were enriched in EMT-related signaling pathways such as PI3K/AKT1 and HIF-1.（2）The results of pathological staining showed that DNA-PKcs deficiency could aggravate lung inflammation after irradiation,resulting in obvious rupture and thickening of lung septum,abnormal expansion of alveolar space and increased exudation of collagen fibers.（3）High EMT was observed in DNA-PKcs deficient mice lung tissue.In vitro and in vivo results showed that DNA-PKcs deficiency promoted Twist expression,suggesting that DNA-PKcs participated in radiation-induced EMT by regulating Twist.（4）DNA-PKcs deletion inhibited the phosphorylation level of SQ/TQ sites on Twist,but these site mutations did not affect the interaction between DNA-PKcs and Twist,suggesting that other proteins may regulate the interaction between them.（5）DNA-PKcs deficiency inhibits AKT1 activity and reduces the interaction between AKT1 and Twist.AKT1 selective agonist IGF-1 can reverse the high expression of Twist and EMT-related protein changes caused by DNA-PKcs deletion.（6）Compared with IR group,VND3207 intervention group had less inflammatory infiltration in the lungs,relatively complete alveolar structure,and a small amount of collagen fiber exudation.（7）VND3207 can increase the self-phosphorylation activity of DNA-PKcs in lung tissue and A549 cells after irradiation in vivo and in vitro,promote DNA damage repair,and promote cell proliferation,which has certain anti-apoptosis and anti-pyroxia effect.（8）VND3207 promotes the expression of E-cadherin and Twist by activating AKT1 through DNA-PKcs.（9）Radiation inhibits the expression of miR-486-3p in A549cells and increases the expression of Snail,thereby promoting EMT.（10）Overexpression of miR-486-3p in vivo can effectively alleviate radiation-induced lung inflammatory infiltration,thickening of lung septum rupture and exudation of collagen fibers.Conclusion:1.DNA-PKcs deficiency aggravates radiation-induced lung injury and lung fibrosis.2.DNA-PKcs/AKT1/Twist axis mediates lung EMT induced by IR and participates in the occurrence and development of RIPF.3.VND3207 activated DNA-PKcs Ser2056 self-phosphorylation activity in vitro and in vivo,reduced DNA double-strand breaks,and promoted the proliferation,anti-apoptosis and anti-pyroptosis ability of lung epithelial cells through DNA-PKcs to alleviate radiation-induced lung injury.4.VND3207 can inhibit the EMT of lung epithelial cells induced by IR through DNA-PKcs/AKT1/Twist axis,and effectively alleviate the occurrence and development of RIPF.5.Radiation-responsive miR-486-3p inhibits lung EMT induced by IR by targeting Snail.The overexpression of miR-486-3p mimics carried by adeno-associated virus（AAV）in lung tissue can effectively alleviate RIPF.