Study On The Regulation Of EZH2 And ATM In Radiation-induced DNA Damage Repair

BACKGROUND:DNA damage caused by ionizing radiation?IR?is one of the important contents of radiation damage effect,and it is also one of the main contents of radiation biology research.The ATM/Chk2/p53 pathway plays an important role in the process of radiation-induced DNA damage repair.ATM is activated immediately after DNA damage,and activates Chk2,phosphorylates p53,or directly transmits damage signals through p53,which opens the process of DNA damage repair.With the deepening of radiation research and the rise of epigenetics,it has been found that histone modifications play an important role in DNA damage repair.Valérie Borde et al.found that after IR irradiation,SPR-5 rapidly aggregated into DNA double-strand breaks?DSBs?to remove H3K4me2,thereby recruiting double-strand break repair?DSBR?proteins.Repair DNA damage.Sheema Fnu et al.found that H3K36me2 in radiation-induced DSB can be repaired by non-homologous end joining?NHEJ?-mediated DSB injury.H3K9me3 interacts with Tip60 in regions of DNA damage in IR-induced mouse embryonic fibroblasts,triggering ATM activation and DNA-damage response?DDR?to repair DSB.At present,H3K27me3 is rarely studied in DNA damage repair.Zhiqing Li et al found that PRC2-mediated H3K27me3 increased in UV-induced DNA damage in silkworm,and it was found that silkworm cells with deletion of PcGs gene showed UV-C radiation highly sensitive.At the same time,ShuBin Gao et al.showed that inhibition of H3K27me3 in HepG2 cells by the EZH2 methyltransferase inhibitor GSK126 promotes DNA damage induced by the chemotherapeutic drug MMC.The above studies confirmed the important role of histone methylation in DNA damage repair.PURPOSE:By establishing EZH2 low-expression and HepG2 over-expression cell model,the role of H3K27me3 in radiation-induced DNA damage was confirmed,and the regulation mechanism of H3K27me3 on ATM/Chk2/p53 by PcGs family member1.The cell cycle of HepG2 cells in different treatment groups was detected by flow cytometry.2.The expression of?H2AX and H3K27me3 in HepG2 cells of different treatment groups was detected by immunofluorescence.3.The expression of EZH2,BMI1,ATM,Chk2 and p53 genes in HepG2 cells were detected by Real-time PCR.The expression levels of EZH2,H3K27me3,ATM and p53 proteins were detected by Western blot.4.Transient transfection method was used to establish the EZH2 low expression and over-expression HepG2 cell model,and observe the regulatory relationship between EZH2 and ATM/Chk2/p53 pathway.5.The co-immunoprecipitation technique was used to detect the modification of H3K27me3 in the ATM promoter region of HepG2 cells in different treatment groups.RESULT:1.Effect of ionizing radiation on HepG2 cell cycle,DNA damage,target gene transcription and protein expressionG₂ phase arrest occurred at doses of 12h and 24h in HepG2 cells irradiated in a dose-dependent manner.The focal numbers of?H2AX and H3K27me3 were significantly increased in each irradiation group compared with the control group,and DNA damage and H3K27me3 expression increase dependent dose.The expression levels of EZH2 and BMI-1 mRNA in the PcGs family members in the radiation group were significantly higher than those in the control group;The expression levels of EZH2 and H3K27me3 proteins were significantly higher in the radiation group;The expression levels of ATM,Chk2,and p53 mRNAs were significantly higher after irradiation.The expression level of ATM and p53 protein was also significantly higher than that of the control group.2.Effect of radiation on cell cycle,DNA damage,target gene transcription and protein expression after inhibition of EZH2 enzyme activityAfter irradiation with different doses of HepG2 cells,2?mol/L GSK126 was added to inhibit EZH2 enzyme activity,and G2 arrest of HepG2 cells occurred in a dose-dependent manner,and G2 arrest was observed in IR+GSK126 group significantly decreased compared with IR group;Immunofluorescence results showed that the number of focal points of H3K27me3 was significantly reduced in IR+GSK126 group compared with IR group,while the focal number of?H2AX was significantly increased;Gene expression results showed that the expression level of EZH2 mRNA in IR+GSK126 group was significantly higher than that in IR group.However,the expression level of H3K27me3 protein was significantly decreased.At the 6th hour after irradiation,the expression levels of ATM and p53 mRNA were significantly increased in IR+GSK126 group compared with IR group,but the expression of Chk2 mRNA was not changed significantly;However,the dose was8Gy after 12h.IR,GSK126 group compared with IR group,ATM,Chk2 and p53mRNA expression levels were significantly reduced.3.Effect of radiation on the expression of ATM,Chk2 and p53 in EZH2interference and over-expressionThe results of immunofluorescence showed that the number of focal points of?H2AX increased significantly in the different irradiation dose groups after interference with EZH2,while the number of focal points of H3K27me3 decreased significantly;the gene expression level results showed that EZH2 mRNA expression level interference irradiation group and the simple irradiation group were significantly decreased.The expression level of H3K27me3 protein was also decreased.The EZH2mRNA and H3K27me3 protein expression levels were significantly increased in the EZH2 over-expression irradiation group compared with the irradiation group.The expression levels of ATM,Chk2,and p53 mRNA were significantly higher in the EZH2 interference group and the irradiation group than in the irradiation group 6 h after the irradiation;12h after irradiation,EZH2 interference group ATM,p53 protein expression levels increased significantly than irradiation group;After EZH2 over-expression,ATM,Chk2 and p53 gene transcription and protein levels did not change significantly.4.Modification of H3K27me3 in ATM promoter region in GSK126 treatment group and EZH2 interference groupWe selected three H3K27me3 binding sites in the-1630^-774 region and 719³475 region downstream of the ATM promoter and detected the expression of H3K27me3 by using the ChIP assay.We found that the siEZH2 and GSK126 groups and the ATM promoter region H3K27me3 The level of expression is significantly reduced.And the expression of H3K27me3 was the most obvious in PP4 loci.CONCLUSION:1.The ATM/Chk2/P53 pathway was activated in ionizing radiation-induced DNA damage response in HepG2 cells.2.EZH2 methyltransferase inhibitor GSK126 promotes ionizing radiation-induced DNA damage by reducing H3K27me3 level of expression.3.The key genes EZH2 participates in DNA damage repair by mediating H3K27me3 in the ATM promoter region.