The Study On The Effect And Mechanism Of Nm23-H1 Mediating DSBs Repair Induced By Ionizing Radiation In A549 Lung Cancer Cells

Introduction:Common malignant tumor of lung cancer mortality in the first place,radiotherapy is one of the main methods of comprehensive treatment of locally advanced lung cancer,but due to radiotherapy central fatal and potentially lethal damage repair caused by radiation resistance reduces the sensitivity of lung cancer radiotherapy to a great extent,so people trying to repair DNA damage for targeted therapy to inhibit tumor cell damage repair.Nm23-H1 as a tumor metastasis suppressor gene,has two nucleoside diphosphate kinase(NDPK)and histidine / serine kinase,also has 3 '-5' exonuclease activity.Therefore,the present study suggests that Nm23-H1 may also be involved in DNA damage repair.Our previous studies and literature reports confirm that Nm23-H1 is related to base excision repair and nucleotide excision repair in DNA damage repair.We also found that Nm23-H1 in ionizing radiation(Ionizing Radiation IR)nuclear translocation induced by ionizing radiation induced DNA double strand breaks(DSBs),so it could also participate in the repair of DSBs damage,and this mechanism has not been reported.Objectives:This study adopt A549 lung cancer cell as the research object,inhibit or nuclear expression of Nm23-H1 protein in A549 cells,exploring the study on the effect and mechanism of Nm23-H1 mediating DSBs repair induced by ionizing radiation in A549 lung cancer cells.Materials and Methods:1.Inducible shNm23-H1 expression vector and a vector with a nuclear localization signal of Nm23-H1 was built in A549 lung cancer cells;Using Western blot test the expression of Nm23-H1 protein,immunofluorescence test nuclear over expression of cell line Nm23-H1 protein subcellular localization;The A549 cell proliferation was assessed using a CCK-8.2.Nm23-H1 detection in ionizing radiation induced lung cancer cells the role of DSBs: using single cell gel electrophoresis experiment test 8 gy X-ray ionizing radiation after 1 h,2 h,4 h fracture damage when double-stranded DNA repair;By confocal laser to detect?H2AX-foci experiments to observe DSBs repair after 8 Gy X-ray ionizing radiation.3.The mechanism of Nm23-H1 mediating DSBs repair induced by ionizing radiation in A549 lung cancer cells : Western blot detect activating ATM and the phosphorylation of its downstream pathway after 8Gy X-ray ionizing radiation;By co-immunoprecipitationn experiment testing DNA damage repair protein combined with Nm23-H1 after 8 Gy X-ray ionizing radiation.Results :1.The Nm23-H1 shRNA and nucleus directional Nm23-H1 lentiviral vector was successfully built.The expression of recombinant plasmid pLentis-Bid-tetO-H1-NM23-SFFV-GTP enzyme digestion and sequencing results of sequencing verified Western blot to verify the stable transfected cell line was constructed;Nm23-H1(NME1)expression vector pLentis-CMV-NME1-IRES2-PURO restriction enzyme digestion and sequencing results were sequenced to verify the correct positioning of the nucleus,and the expression of Western was located mainly in the cytoplasm of blot A549 cells without transfection verified Nm23-H1 protein,and the transfection group Nm23-H1 protein in cytoplasm and nucleus were detected,and the expression mainly in nucleus.Immunofluorescence test showed that the Nm23-H1 protein in the non transfected group was mainly located in the cytoplasm of A549 cells,and the expression of the protein increased significantly in the nucleus after transfection.CCK-8 method was used to detect Nm23-H1 nuclear cells proliferation,the results showed that compared with empty vector group and blank control group,no significant difference between the two,Nm23-H1 nuclear expression group proliferated faster than control group and empty vector group,compared with the empty vector group was significant difference in 72 h,96 h and 120 h.2.The study on the effect of Nm23-H1 mediating DSBs repair induced by ionizing radiation in A549 lung cancer cells.(1)Single cell gel electrophoresis experiment results show that the A549 cells after 8 Gy X-ray irradiation,knock Nm23-H1 low group compared with control group,the low group can significantly increase the tail DNA content,prompt Nm23-H1 fracture damage may participate in DSBs repair;Nm23-H1 nuclear express group shows its in 1 h,2 h,4 h was a comet tail moment significantly reduced,and it has significant difference(P < 0.05),the same tip Nm23 H1-participate in double-stranded DNA damage repair.(2)Laser confocal detection ?-H2 AX foci,according to the results of A549 cells after 8 Gy X-ray irradiation,Nm23-H1 knockdown group over time?-H2 AX foci positive cells number dissipation was slow,at 8 h difference has significant difference(P < 0.05);Nm23-H1 nuclear express group of?-H2 AX foci positive cells number after 2 hr dissipate faster than the control group(P < 0.05),and the results are on the low group is more apparent,prompt Nm23-H1 participated in the repair DSBs caused by ionizing radiation damage,and this effect may be associated with Nm23-H1 nuclear transfer.3.The study on the mechanism of Nm23-H1 mediating DSBs repair induced by ionizing radiation in A549 lung cancer cells.(1)By Western blot detection after 8 Gy X-ray ionizing radiation ATM activation of the phenomenon,and detect its molecular pathways downstream of the phosphorylation level of change,the results show that Nm23-H1 nuclear expression cell group after 8 Gy X-ray irradiation can make pS1981-ATM ? pS15-p53 ? pT68-Chk2 expression are enhanced,while Nm23-H1 knock the low group these three kinds of phosphorylated proteins expression even less does not mean that Nm23 H1-can express through the ATM?p53?Chk2 phosphorylation activation involved in repair DSBs damage.(2)By immune coprecipitation experiments,testing 8 Gy X-ray ionizing radiation Nm23-H1 combined with protein involved in DNA damage repair,the results showed 8 Gy X-ray irradiation,after 1 hr in Nm23-H1 immune precipitation compounds detected NBS1 RAD50 MRE11 Ku80 proteins,especially NBS1 and Ku80 protein expression in Nm23 H1-nuclear nuclear group is more obvious;When lung cancer cells by ionizing radiation,is not in the precipitation Nm23-H1 immune complexes the protein that Nm23-H1 can be detected through the combination of proteins associated with DNA damage repair to participate in the repair DSBsConclusion :1.Nm23-H1 can induce the nuclear plant to establish directional on expression of Nm23-H1 low A549 cell lines and we first constructed,which lays the foundation for further study of Nm23-H1 involved in the repair of DNA double strand breaks,and found that Nm23-H1 nuclear overexpression promotes cell proliferation of A549 cells.2.Single cell gel electrophoresis and?-H2 AX foci detection results showed that Nm23-H1 was involved in the repair of DNA double strand breaks in A549 lung cancer cells induced by ionizing radiation.3.Nm23-H1 may through two mechanisms involved in DSBs damage repair,one is the activation of the ATM-Chk2-p53 pathway involved in DSBs damage repair by phosphorylation;one is through a combination of DNA damage repair proteins,especially in combination with NBS1 and Ku80 in DSBs damage repair.