Role And Mechanism Of PC4 Regulated DNA Repair Via Non-homologous End Joining In Radiosensitivity In Non Small Cell Lung Cancer

Objective: To clarify the effects of Human positive cofactor 4(PC4)-regulated DNA repair via non-homologous end-joining(NHEJ)in radiosensitivity of non small cell lung cancer(NSCLC),and to investigate the potential mechanism involved in this process.Methods: Expression of PC4 was first examined in four NSCLC cell lines by western blot,and A549 and PC-9 were then selected as models.Recombinant lentivirus was obtained by co-transfection of HEK 293 FT cells,and infected A549 and PC-9 cell lines.After selection by puromycin,the clones were analyzed for PC4 knockdown(sh PC4),PC4 restoration(sh PC4+PC4)or control(Mock).Transfectants were irradiated with 0,2,4,6 and 8Gy X-ray.Cell proliferation and apoptosis was determined by colony formation assay and FACS analysis.The levels of PC4,XLF,XRCC4 and DNA ligase IV were examined by western blot and PCR assay.And location of XLF and DSB and their effects on DNA repair were analyzed by immunofluorescence assay.Interaction between PC4 and XLF were measured by immunoprecipitation and chromosome immunoprecipitation.Results: Expressions of PC4 were higher in NSCLC cell lines,including H1975,H460,A549 and PC-9,compared with normal pulmonary epithelial cells,Beas-2B.Knockdown of PC4 significantly suppressed colony formation and promoted radiosensitivity in A549 and PC-9 cells,and vice versa.Furthermore,silenced PC4 also decreased cellular apoptosis.Besides,PC4 downregulation inhibited expression of XLF and interaction between PC4 and XLF,while expressions of XRCC4 and DNA ligase IV didn’t change.Immunofluorescence assay showed that upon irradiation,expression of XLF and DSB repair in PC4 silenced cells,and consistently,higher PC4 resulted in restoration of XLF expression and DSB repair.Results of IP and CHIP showed that PC4 regulated transcription of XLF,and downregulated PC4 suppressed interaction between PC4 and XLF.Conclusion: Depletion of PC4 inhibits XLF transcription,and increases radiation-induced cellular apoptosis by suppressing DNA repair via NHEJ.This mechanism is related to higher radiosensitivity of NSCLC cells.PC4 may regulate DNA repair via non-homologous end joining in radiosensitivity in non small cell lung cancer.