| Lung cancer is one of the malignant tumors with the highest rate of incidence and mortality growth thatpose aserious threat to health,ranking the first place of human cancerous death.Studies have shown that abnormal histone methylation are closely related to tumor occurrence and development.Histone methylation is an important epigenetic modification that plays an important role in the regulation of gene expression.Radiation resistance of tumor cells is one of the important reasons why tumors can not be completely controlled during radiotherapy.Therefore,exploring the relationship between lung cancer cell radiation resistance and histone methylation modification of lung cancer radiation therapy has important guiding significance.Background: The mixed lineage leukemia(MLL)family of genes,also known as the lysine N-methyltransferase 2(KMT2)family,consists of six members(MLL1,MLL2,MLL3,MLL4,SETD1 A and SETD1B).All of members help to regulate the transcriptional activation of downstream genes.Studies have shown that the MLL family of proteins is highly specific for histone H3 lysine 4.Recently,it was found that MLL5 exerts the effect of histone methyltransferase by regulating the expression of other histone modifying enzymes such as LSD1 or SET7 / 9.The reduced rate of H3K4 trimethylation of the E2F1 target promoter in MLL5 knockdown cells suggests that MLL5 interacts with other proteins such as HCF-1 to indirectly stimulate methylation.It has been reported that MLL5 plays a key role in a variety of biological processes including cell cycle progression,maintenance of genomic stability,adult hematopoiesis,and spermatogenesis.EZH2(enhancer of zeste homolog2)is the only subunit with enzymatic activity in PRC2 complex,but SUZ12,EED and YY1 are indispensable components of PRC2 enzyme activity.As an important epigenetic transcriptional repressor,PRC2 complex plays an important role in embryonic development,stem cell maintenance,X chromosome inactivation,tumorigenesis and metastasis.EZH2,a member of PRC2 complex,plays a role in gene regulation by stimulating the activation of histone methyltransferase 27 of histone H3 at lysine 27,and PRC2 complex influences gene regulation through its own interactions.In this research,we studied the expression changes of MLL5 and EZH2 and their respective modified histone methylation levels in lung cancer cells exposed to radiation and emerged radiation resistance,as well as exposing the regulatory patterns of MLL5 complex and EZH2 downstream genes,to clarify the function and mechanisms of MLL5 and EZH2 in regulating histone modifications in radiation resistance.Purposes: 1.To research the regularity of MLL5 and EZH2 and its regulation of histone methylation in lung cancer cells subjected to single exposure and the procedure of emerging radiation resistance;2.To investigate the regulation of histone methylation by MLL5 and EZH2 after single exposure and radiation resistance in lung cancer cells.3.To investigate the effect of MLL5 and EZH2 on cell cycle progression in radiation resistance of lung cancer cells.Methods: 1.A549-RR model was constructed by the method of fractionated high-dose(6 Gy/times)irradiation,that is A549 cells continued to be cultured for 10-12 days after each irradiation,and they were exposed to 6 Gy after the cells were stable,and repeated.When the cumulative dose reached 30 Gy,cell clone formation assay and MTT assay were used to test whether the A549-RR model was successfully established.2.The si MLL5-A549,si EZH2-A549,si MLL5-A549-RR and si EZH2-A549-RR cell models were constructed by transient transfection of si MLL5 and si EZH2 into A549 and A549-RR cellsrespectively.3.Real-time PCR was used to detect the changes of transcription levels of MLL5 and EZH2 at different time after irradiation and in theformation process of radiation resistance.4.Western Blot method was used to detect the change of protein levels of MLL5 and EZH2 and the levels of H3K4me2,H3K4me3 and H3K27me3 at different timeafter single irradiation and in theformation process of radiation resistance;5.Flow cytometry was used to detect changes in the cell cycle progression of A549 cellsand A549-RR cell model that interfered with MLL5 and EZH2 genes.Results: ?.Establish a variety of cell models The A549-RR model was constructed using fractionated high-dose(6 Gy/times)irradiation,that is A549 cells continued to be cultured for 10-12 days after each exposure,they were exposed to 6 Gy after the cells were stable,and repeated.Accumulative doses At 30 Gy,the cell clone formation assay and MTT assay were used to test whether the A549-RR model was successfully established.The results showed that compared with wild-type A549,the colony-forming ability and proliferation ability of radiation-resistant cells were significantly increased(p<0.05).Transient transfection method was used to transfect si MLL5 and si EZH2 interference fragments into A549 cells.The transcription and protein levels of MLL5 and EZH2 were detected by Real-time PCR and Western blot,respectively.The results showed that the transcription level of MLL5 gene in the si MLL5-A549 group was 0.46 times that of the control group,and the transcription level of the EZH2 gene in the si EZH2-A549 group was 0.65 times that of the control group,and all had statistical significance(p< 0.01).Western blot results showed that MLL5 and EZH2 protein were significantly downregulatedin MLL5 and EZH2 si RNA transfection group compared with the control group,the degree of declining was 0.25 and 0.23 times,indicating that the two gene interference cell model was successfully constructed.?.Role of MLL5 in radiation-induced histone methylation modification in lung cancer cells 1.Effect of ionizing radiation on MLL5 and H3K4me3/2 in A549 cells Real-time PCR results showed that the wild-type A549 was given a single 10 Gy X-ray exposure,and the MLL5 m RNA expression level transiently decreased at 3 h after exposure(p< 0.001)but increased at 24 h.(p< 0.001).Western blot results showed that the protein expression of MLL5 increased at 3 h after exposure,but decreased significantly at 24 and 48 h.Western blot analysis showed that the expression of H3K4me3 in A549 cells was basically decreased after single exposure,and decreased significantly after 10 minutes,and decreased to 0.2 times in 48 hours.The change of H3K4me2 did not change significantly 24 hours or less after irradiation,but its expression decreased to 0.3 times that of the control group 48 hours after irradiation.Real-time PCR showed that the transcription level of MLL5 was not stable after the first four irradiationsduring the process of constructing the A549-RR model,and was significantly decreased after the first and fourth irradiation(p< 0.001).After last irradiation(30 Gy cumulative dose),the transcription level of MLL5 increased to 3.37 times that of the control group(p< 0.001).Western blot results showed that the change of MLL5 protein was more stable,is higher than that of the control group after each irradiation.When the radiation resistance was completely formed(30 Gy cumulative dose),it was still nearly 2 times higher than that of the control group.After irradiating with 10 Gy X-rays,protein co-immunoprecipitation was used to detect the binding of NRP1 and MLL5 proteins.The results showed that after A549 cells were exposed to a single irradiation,the binding of NRP1 to MLL5 protein increased,but the protein binding was significantly reduced after acquring radiation resistance.It is suggested that although single exposure and radiation resistant A549 cells increase the expression of NRP1(as confirmed in previous studies),the strength of binding ability to MLL5 plays an important role in the development of radiation resistance in lung cancer cells.The expression of H3K4me3 detected by western blot showed that when A549-RR cells model were constructed,its expression was significantly lower than that of the control group after the first three irradiations(radiation resistance was not completely produced),and gradually increased from the fourth time to the last irradiation(the cumulative dose reached 30 Gy)and the increase was 2.11 times.Changes in H3K4me2 increased slightly after each irradiation,but there was no significant change.The results showed that MLL5 and its regulatory H3K4 methylation appeared opposite results during the single exposure and radiation resistance of lung cancer cells,and H3K4me3 was the main modification,but no significant relationship with H3K4me2 modification.2.Role of MLL5 in the regulation of H3K4 methylation in single-irradiation and radiation-resistant lung cancer cells A549 cellsinterfere withthe expression of MLL5 gene plus a single irradiation,H3K4me3 expression was significantly reduced(0.2 times that of the control group),while the expression of H3K4me2 was not significantly related to the change of MLL5.The expression of H3K4me3 was significantly increased in A549-RR cells,although it decreased after interference with si MLL5 fragment,but it was still higher than that of the control group.3.Radiation-induced modulation of MLL5 on its recruited complexes and downstream target genes Compared with the control group,the levels of MLL5,E2F1 and HCF-1 m RNA were significantly lower in the group irradiated alone,while OGT was significantly higher(p< 0.001).After MLL5 gene disruption,OGT,USP7,E2F1 and HCF-1 allN decreased significantly,and both were statistically significant.On this basis plus asingle 10 Gy X-ray exposure,the m RNA levels of OGT and HCF-1 were significantly increased(p< 0.01).The detectionresults of the target genes downstream of MLL5 showed that the HOXA9 and Cyclin A genes in the group irradiated only were significantly lower(p< 0.001).After interfering with MLL5 gene irradiation,the m RNA level of Cyclin A decreased more significantly,and the m RNA level of HOXA9 increased significantly(p< 0.001).This indicates that MLL5 directly regulates HOXA9 in single irradiation of lung cancer cells,thereby affecting histone methylation modification..The expression levels of MLL5 and OGT in A549-RR cells were significantly increased(p < 0.001),and there was no significant change in USP7,E2F1 and HCF-1.After transfected with si MLL5 in A549-RR cells,MLL5 decreased,E2F1 and HCF-1 expression increased(p < 0.001),and OGT and USP7 had no significant changes.This phenomenon was different from that after single irradiation.The detection results of downstream target genes of MLL5 showed that the transcription level of HOXA9 in A549-RR cells was significantly increased(p< 0.001),but there was no significant change in Cyclin A.There was no significant change in Cyclin A and HOXA9 genes in A549-RR cells that interfered with MLL5 gene.?.Role of MLL5 in radiation-induced histone methylation modification in lung cancer cells 1.Effects of ionizing radiation on EZH2 and H3K27me3 in A549 cells Real-time PCR results showed that the EZH2 m RNA levels in wild-type A549 cells decreased in a time-dependent mannerafter a single 10 Gy X-ray irradiation,and they were down-regulated to 0.44 times(p< 0.001)at 48 h.Western blot results showed that EZH2 protein levels began to decrease at 12 h after exposure,and reached to 0.45 times in the sham group at 48 h.After a single irradiation,the expression of H3K27me3 in A549 cells was transiently reduced(within 1 h after a single radiation),then the expression level of H3K27me3 was gradually restored to that of the control group.In the process of emerging radiation resistance in A549 cells,there was no regular change in m RNA levels of EZH2,when the radiation dose accumulated to 30 Gy(radiation resistance had been generated),and the expression of EZH2 gene was significantly increased,which was 3.91 times higher than that of the control group.(p< 0.001).Western blot results showed that EZH2 protein expression was not reduced regularly in the process of radiation resistance,but EZH2 protein expression was increased about twice as much as that of the control group at a cumulative irradiation dose of 30 Gy(radiation resistance had been generated).In the process of emergingradiation resistance of A549 cells,the expression level of H3K27me3 after each exposure was higher than that of the control group,and its expression level was about 8 times higher than that of the control group,compared with the expression of H3K4me3(2.11 fold),it is 4 times higher.Therefore,although MLL5 and EZH2 have a positive regulation on H3K4me3 and H3K27me3,respectively,the modulation of EZH2 on H3K27me3 is mainly responsible for the radiation resistance of lung cancer cells..2.Regulation of EZH2 on H3K27me3 in single-irradiation and radiation-resistant lung cancer cells The results of Western blot showed that the expression of H3K27me3 was down-regulated to 0.3 times that of the control group after interfering with EZH2 gene.On this basis plus a single radiation,the H3K27me3 reduction was more significant,about 0.1 times that of the control group(p< 0.001).The level of H3K27me3 was significantly increased after radiation resistance was induced in A549 cells,but it was significantly decreased after interference with EZH2(p< 0.001).3.Radiation-induced regulation of downstream target genes by EZH2 Real-time PCR results showed that the EZH2-regulated downstream target gene p21 increased to 4.21-fold and p27 decreased to 0.80-fold after a single irradiation with 10 Gy X-rays,with statistical significance(p< 0.001).However,when EZH2 gene was interfered plus a single irradiation,EZH2 decreased more significantly,and p21 increased more significantly(4.46 times,p< 0.001),while p27 transcription level was significantly higher than that of the simple irradiation group(p< 0.01),this indicates that EZH2 does not directly regulate the expression of downstream genes in a single exposure,and may regulate H3K27me3 modification through another mechanism.Compared with wild-type A549,EZH2,p21 and p27 genes were up-regulated in A549-RR cells(both were about 2 fold higher than in the control group).The expression of p21 and p27 in the A549-RR cells that interfered with the EZH2 gene also increased,but was even more significant(p< 0.001),about 3 times of the control group.Obviously,single-irradiation and repeated irradiation lead to radioresistance in lung cancer cells,EZH2 regulation of downstream genes is not exactly the same.?.Effect of MLL5 and EZH2 on phenotype of irradiated A549 cells 10 Gy single irradiation can induce G2/M phase arrest in A549 cells,while A549-RR cells have S phase prolongation;It still showed that G2/M phase arrest in A549 cells after single MLL5 gene interference,and the S phase of the cells was significantly shortenedin A549-RRcells,with G1 and G2/M arrests.It is suggested that there are different cell cycle changes in single exposure and radiation-resistant lung cancer cells,but there is no direct relationship with the regulation of the downstream gene Cyclin A,which needs to be further explored.When the EZH2 gene was interfered plus a single irradiation,S phase shortened and G2/M phase arrest occurred,whereas S phase prolonged,and G1 phase shortened of A549-RR cells.This suggests that the mechanisms of action of the two histone methyltransferases on cell cycle progression are different,and in particular,there has been no G1 arrest in radiation-resistance lung cancer cells,and this result is consistent with the result that EZH2 does not regulate p21 directly.Conclusions: 1.The expression of MLL5 and EZH2 protein was inhibited after a single exposure of A549 cells,but its expression was up-regulated after emerging radiation resistance.It may be related to the different binding capabilities of NRP1 and MLL5.2.MLL5 can inhibit the recruitment of OGT/HCF-1 complex and modulate the downstream target gene HOXA9 in a single irradiation of A549 cells;MLL5 inhibits the recruitment of E2F1/HCF-1 and does not activate downstream target genes after emergingradiation resistance.;3.EZH2 only regulates the downstream gene P27 in single-irradiated lung cancer cells,but negatively regulates downstream genes P21 and P27 in radiation-resistant cells.4.MLL5 influences radiation-resistant cells by shortening S phase and causing G1 phase and G2/M phase block,whereas EZH2 exerts influence by extending S phase.5.In the radiation resistance of lung cancer cells,MLL5 and EZH2 relatively regulate the modification of histone methylation,and the effect of H3K27me3 is more significant than that of H3K4me3. |
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