EZH2 Regulates ATM/p53 Pathway In Inducing DNA Damage Repair Of Hep3B Hepatoma Cells By Ionizing Radiation

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world.About 38.3 million people die of liver cancer each year in China,accounting for 51% of the world's liver cancer deaths.The World Health Organization had expected that according to the current rate of increase in cases,approximately 10 million people will die from cirrhosis and liver cancer from 2015 to 2030 in China.Actively seeking effective treatment of liver cancer and improving the feasibility of treatment are the primary problems to reduce the mortality of liver cancer at the current stage.Radiotherapy is a commonly used method for treatment of cancer,especially for the treatment of nasopharyngeal and lymphoma.The radiotherapy for skin cancer and esophageal cancer can basically achieve the therapeutic effect.The United States National Comprehensive Cancer Network(NCCN)guideline for the diagnosis and treatment of liver cancer has been recommended since 2013: Patients with primary liver cancer are suitable for external exposure regardless of where the tumor is located.Liver cancer radiotherapy is also recommended in the guidelines for the diagnosis and treatment of liver cancer in China.The resistance of cells to ionizing radiation largely limits the therapeutic effects of tumors.Although in recent years,many inhibitors or activators that target radiation-related molecules have been developed as radiation sensitizers,the radiation resistance of cells is a complex and sophisticated self-healing network targeting a single molecule.Drug design does not completely eliminate the compensatory mechanisms of cells.Therefore,finding the switch molecules that have a direct relationship with cell radiation resistance in the cell self-repairing mechanism is the key to solving the radiation resistance problems commonly encountered in the current stage of liver cancer radiotherapy.Research Background:The most serious biological damage to cells caused by ionizing radiation is DNA double-strand breaks(DSBs).After DSBs occur,the body rapidly activates DDR,blocking cell cycle progression and providing time protection for damage repair.At the same time,the DSBs site will accumulate multiple repair proteins to repair broken DNA sites.This mechanism is thought to have an important link with the radiation resistance of tumor cells.The ATM gene is an important cell cycle checkpoint kinase that rapidly recognizes DSBs and,once activated,initiates a series of phosphorylation cascades that regulate downstream genes such as p53,MDM2,Chk2,NBS1,RAD9,and BRCA1 and participate in regulation.Cell cycle progression,participation in DDR.The ATM/Chk2/p53 pathway has been widely reported.The polycomb group protein complexes(PcG)protein complex mediates the target gene methylation modification to achieve transcriptional inhibition of the target gene and participate in the regulation of various biological functions of the human body.PcG consists of two core protein complexes: Polycomb repressive complex 1(PRC1)and Polycomb repressive complex 2(PRC2).More and more experiments have shown that PcG is involved in mammalian DDR regulation in addition to regulating stem cell differentiation and cancer occurrence and progression.PRC2 mainly exerts the trimethylation of histone 3 lysine 27(H3K27me3)catalysis.EZH2,as a catalytic subunit of PRC2,is a histone methyltransferase that directly participates in the modification of H3K27me3.SUZ12 and EED mainly function as stable complexes.It is considered that H3K27me3 is a marker of PcG inhibiting transcription of a target gene.Summarizing existing research results,it can be determined that when DNA double-strand breaks occur in cells,PcG is recruited at the DSBs site and may participate in DNA damage repair,but the specific regulatory mechanisms are rarely reported.Research purposes:1.When ionizing radiation induces DNA damage in Hep3 B cells,the expression of EZH2 and BMI-1 in PcG increases;2.When the DNA damage of Hep3 B cells was induced by ionizing radiation,the expression level of each gene in ATM/Chk2/p53 pathway was increased;3.Decreased EZH2 expression level,enhanced Hep3 B radiation sensitivity;4.EZH2 regulates ATM expression by mediating H3K27me3 in the promoter region of ATM,thereby realizing the regulation of ATM/Chk2/p53 pathway.Research methods:1.The CCK8 method was used to detect the growth inhibitory effect of Hep3 B cells under the action of EZH2 inhibitor GSK126.The appropriate dose of GSK126 was selected for subsequent experiments;2.The expression of ? H2 AX and H3K27me3 in irradiated Hep3 B cells was detected by immunofluorescence.It was verified that ionizing radiation caused DNA double-strand breaks in Hep3 B cells,and DNA damage and H3K27me3 expression increased with the increase of radiation dose.Explore the relationship between EZH2 and DNA damage.3.Flow cytometry was used to detect the change of cell cycle after different doses of ionizing radiation in IR+GSK126 group and IR group Hep3 B cells.It was confirmed that ionizing radiation can cause cell cycle arrest,and H3K27me3 of EZH2 is involved in the regulation of cell cycle arrest.4.Hep3B-EZH2 low and Hep3B-EZH2 high cell models were established using transient transfection method to investigate the regulatory relationship between EZH2 and ATM,Chk2,p53.5.Real-time PCR and Western Blot methods were used to detect the expression levels of EZH2,BMI-1,ATM,Chk2,and p53 mRNA and protein in each cell model line,to explore the role of these genes in the regulation of radiation resistance of Hep3 B cells,and EZH2 The regulatory relationship with ATM,Chk2,p53.6.Chromatin immunoprecipitation(Ch IP)technique was used to detect H3K27me3 in the promoter region of ATM,and EZH2 was used to regulate the ATM/Chk2/p53 pathway through the action of H3K27me3.Research result:1.Hep3 B cells were treated with 0.5 Gy,1 Gy,2 Gy,4 Gy,and 8 Gy of 5 different doses of ionizing radiation to detect changes in cell cycle.The results showed that compared with the sham group(0Gy group),the proportion of cells in the G2 phase increased,and the proportions of cells in the G1 and S phases showed different degrees of reduction,and the return showed a significant dose-dependent increase;GSK126 was added.After inhibiting the biological function of EZH2,it was observed in 5 different irradiation dose groups that the G2 arrest in IR+GSK126 group was lower than that in IR group at the same irradiation dose.2.The results of immunofluorescence showed that when Hep3 B cells were irradiated with 4Gy and 8Gy respectively,the expression of ? H2 AX in the cells was significantly higher than that in the sham group(0G group),and the expression of H3K27me3 was also significantly higher than that of the control group.The expression levels of ? H2 AX and H3K27me3 were dose-dependent;GSK126 inhibited the biological function of EZH2 in Hep3 B cells,and they were irradiated with 4Gy and 8Gy respectively.The results showed that the expression of ? H2 AX in IR+GSK126 group was higher than that in IR group and IR+ The expression of H3K27me3 in the GSK126 group was higher than that in the IR group,which was consistent with the results observed when the same irradiation conditions were given after silencing the EZH2 expression in Hep3 B cells;3.When Hep3 B cells were treated with 4Gy and 8Gy ionizing radiation,EZH2,ATM,and p53 mRNA and protein levels,Chk2,BMI-1 mRNA levels and H3K27me3 protein levels were higher than those in the control group;GSK126 was added to inhibit Hep3 B cells EZH2 After biological function,compared with the control group(Hep3B cells without GSK126),P53 mRNA and protein expression levels did not change significantly,but ATM m RNA and protein expression levels increased;in the EZH2-silenced Hep3 B cell model(Hep3B-SiEZH2),Hep3B-siEZH2 group P53 and ATM mRNA and protein expression levels were higher than the control group(non-transfected Hep3 B cells);in the EZH2 overexpression cell model(Hep3B-overexpression EZH2),Hep3B-overexpression Compared with the control group(non-transfected Hep3 B cells),the expression levels of P53 and ATM mRNA and protein in EZH2 group were not significantly changed;4.ChIP results showed that the expression of H3K27me3 in the six DNA fragments of the ATM promoter PP1-6,Hep3B-siEZH2 group and GSK126-expressed Hep3 B cells was significantly lower than that of the untreated group,and both were PP4 fragment changes are most obvious.Conclusion1.Ionizing radiation can induce DNA double-strand breaks and cell G2 arrest in Hep3 B cells;2.ATM/Chk2/P53 pathway involved in ionizing radiation-induced DNA damage in Hep3 B cells;3.EZH2 and BMI-1 in PcG family are involved in ionizing radiation-induced DNA damage response in Hep3 B cells,and EZH2 expression is decreased,which can increase the radiosensitivity of Hep3 B cells.4.When ionizing radiation induces DNA damage in Hep3 B cells,EZH2 regulates the ATM/Chk2/P53 pathway by mediating the H3K27me3 modification in the ATM promoter region,which in turn influences the radiation effects on Hep3 B cells.