The Function And Mechanism Of AMPKα1 In DNA Double-strand Break Repair Of Ovarian Cancer Cells

Part 1 Whether AMPKα is involved in intracellular DNA double-strand break repair[Purpose] To investigate whether AMPKα is involved in intracellular DNA double-strand break repair.[Methods] This section of the experiment is divided into the following five parts.1.Chromatin components of human ovarian cancer cells SK-OV-3 and HO-8910 as well as human breast cancer cell lines were isolated after DNA double-strand breaks were induced by X-ray treatment.The level of AMPKα in chromatin components before and after induction was detected by Western blot;2.Construct the plasmids of GFP-AMPKα1 and GFP-AMPKα2 as well as the different domains of GFP-AMPKα1 and transfect them into SK-OV-3.After laser treatment of cancer cells,observe whether GFP fusion protein can be recruited to laser-induced DNA damage sites;3.Transfect the plasmid of GFP-AMPKα1into cells,and then use ATM inhibitor ku-55933 and PARP inhibitor ABT-888 to treat the cells,respectively.After DNA double-strand breaks induced by laser treatment,we can observe the recruitment of AMPKα1 at DNA damage sites using fluorescence microscope;4.Construct GFP-AMPKα1,Flag-SBP-AMPKα1,Flag-PARP1 and HA-PARP4 plasmid and transfect them into human embryonic kidney cell 293(HEK293T).HEK293 T were treated with X-ray and H2O2 to cause DNA double-strand breaks,and then protein samples were collected for detecting the interaction between AMPKα1 and PARP1 by Co-Immunoprecipitation;5.Use different concentrations of Bleomycin(BLM0μM,2μM,4μM,6μM,8μM,10μM)to treat SK-OV-3 for 2 hours to induce DNA damage.After 48 hours,the survival rate of SK-OV-3 was detected by CCK8 kit.Based on the known half lethal concentration of BLM,SK-OV-3 was treated with it for 2 hours,and then replaced with normal culture medium for cell repair.Protein samples were collected at different time points for detecting the levels of AMPKα and its phosphorylation by Western blot.[Results] 1.After X-ray treatment of different cancer cell lines,Western blotting detected an enhanced level of AMPKα in chromatin components following DNA double-strand breaks;2.Both AMPKα1 and AMPKα2 can be recruited to DNA damage sites after laser induced DNA double-strand breaks in ovarian cancer cells,and the catalytic domain of AMPKα1 mainly mediates its recruitment to DNA damage sites;3.Treatment with PARP inhibitor ABT-888 resulted in impaired recruitment of AMPKα1 to DNA damage sites,while ATM inhibitor KU-55933 did not affect this process;4.Results of Co-immunoprecipitation assay showed that the interaction between PARP1 and AMPKα1was enhanced after DNA damage;5.According to the CCK8 results,the half lethal concentration of BLM was determined to be 3.3μM.After treatment with BLM 4μM for 2hours,Western blot showed that the phosphorylation level of AMPKα increased.[Conclusions] Both AMPKα1 and AMPKα2 are involved in DNA double-strand break repair in ovarian cancer cells.PARP1 regulates the recruitment of AMPKα1 to DNA damage sites,and the catalytic domain of AMPKα1 is crucial to this process.Moreover,the phosphorylation of AMPKα may participate in the regulation of DNA damage response in ovarian cancer cells.Part 2 Effects of PRKAA1 knockout on DNA double-strand break repair in ovarian cancer cells[Purpose] To establish PRKAA1 knockout model and explore the effects of PRKAA1 knockout on DNA double-strand break repair in ovarian cancer cells.[Methods] This section of the experiment is divided into three parts.1.Use CRISPR-Cas9 to knock out PRKAA1 in SK-OV-3 and human cervical cancer cells He La;2.Use BLM 4μM to treat cancer cells for 2 hours to induce DNA damage and then replaced with normal culture medium for cell repair.Protein samples of the control group and the knockout group were collected at different time points.The phosphorylation levels of ATM and H2 AX were detected by Western blot.The difference of H2 AX phosphorylation levels between the two groups was compared by immunofluorescence;3.Transfect GFP-AMPKα1 into the knockout cells and collect protein samples at different time points after BLM treatment.Western blot was used to observe whether the transfection can rescue this phenotype.[Results] 1.Western blot results showed that PRKAA1 was successfully knocked out in SK-OV-3 and He La,respectively;2.The result of Western blotting showed that compared with the control group,the phosphorylation levels of ATM and H2 AX were significantly decreased in the knockout group,indicating that ATM activation was blocked and the ability of DNA double-strand break repair was impaired.Furthermore,immunofluorescence exhibited the reduced number of γH2AX foci in the knockout group;3.Compared with the knockout group,the transfection of GFP-AMPKα1 increased ATM phosphorylation level and the ability of DNA double-strand break repair of cancer cells.[Conclusions] PRKAA1 deficiency impaired DNA double-strand break repair and ATM activation in ovarian cancer cells,while restoration of AMPKα1 expression can rescue this phenotype.Part 3 The molecular mechanism of AMPKα1 involvement in DNA double-strand break repair[Purpose] To explore how AMPKα1 is recruited to DNA damage sites and influences ATM activation.[Methods] This section of the experiment is divided into two parts.1.Transfect GFP-N1,GFP-AMPKα1 and GFP-AMPKα1 plasmids into three plates of HEK293 T,respectively.The third group was treated with 10μM BLM for 3h to induce DNA double-strand breaks.The first and second groups were treated with the same amount of DMSO for 3h as controls.Protein samples of three groups were collected for Immunoprecipitation and Silver staining as well as Mass spectrometry;2.Flag-AMPKα1and GFP-Artemis,GFP-AMPKα1,Flag-AMPKα1 were transfected into HEK293 T,respectively,and the corresponding empty vector was transfected as the control group.The cells were treated with ICRF or BLM 10μM for 3h to induce DNA double-strand breaks,and then the protein samples were collected.The interactions between AMPKα1 and DNA double-strand break repair-related proteins such as Artemis,BRACA1,RAD50 were detected by immunoprecipitation assay.[Results] 1.The result of Sliver staining showed that compared with the untreated group,numbers of proteins interacted with AMPKα1 significantly increased after 10μM BLM treatment.Then we performed bioinformatics analysis on the proteins identified by mass spectrometry and found that there are some proteins related to the involvement of AMPKα1 in DNA double-strand break repair such as UBR5,PARP1,RAD50,BRCA1/2and Artemis,etc.;4.Results of Co-immunoprecipitation assay showed that the interactions between AMPKα1 and Artemis,BRCA1,RAD50 did exist.[Conclusions] Co-Immunoprecipitation assay combined with mass spectrometry found that AMPKα1 interacts with several proteins involved in DNA double-strand break repair,including Artemis,BRCA1,and RAD50.