The Role Of EZH2 Protein In Regulation Of TGF-β Signaling Pathway In ATL Cells And Its Molecular Mechanisms

Human T-cell leukemia virus type 1(HTLV-1)is a carcinogenic retrovirus that can cause adult T-cell leukemia(ATL).happened.In HTLV-1,it mainly encodes a series of regulatory proteins such as p30,p13,Tax and HBZ.The HTLV-1 virus promotes the development of ATL by regulating multiple signaling pathways including TGF-β.The continuous activation of TGF-β signaling pathway is very important in ATL,especially in the process of viral infection,TGF-β can reduce the immune monitoring function of normal lymphocytes,thus contributing to the continuous infection of the virus and the survival of infected cells;At the same time,TGF-β signaling pathway also plays a key role in virus replication and infection.At present,the molecular mechanism of the continuous activation of TGF-β signaling pathway in ATL cells is not well understood.Therefore,it is extremely important to study the regulatory factors of TGF-β signaling pathway that are continuously activated in ATL cells.The human homolog of Zeste gene enhancer 2(EZH2)is highly expressed in various tumors,and it has been reported that EZH2 participates in the functional regulation of Smad and other proteins in other tumors,and we pass the pre-It was found that the expression level of EZH2 was higher in ATL cells.Then,is EZH2 highly expressed in ATL cells related to the continuous activation of TGF-βsignaling pathway? In order to solve this problem,this topic will be studied in the following aspects:Part Ⅰ: High expression of EZH2 in ATL and its mechanismWe first detected the expression of EZH2 and Smad3 genes in ATL cell line by RT-PCR.We found that EZH2 gene was highly expressed in different degrees compared with the control group(MOLT-4,Jurkat,CEM-T4),ATL cell lines(MT-4,TL-Om1,ATL-T,ATL-2,MT-1,MT-2).Western blot also showed that the expression of EZH2 protein in HTLV-1 positive cell line was significantly higher than that in negative cell line.In order to further study the molecular mechanism of high expression of EZH2 in ATL cell line,we carried out double luciferase reporter gene.The results showed that the Tax protein encoded by HTLV-1 could activate the promoter activity of EZH2.Western bolt assay further confirmed that overexpression of Tax protein in T cells could promote the high expression of EZH2 protein.Part Ⅱ: The regulation of TGF-β signaling Pathway by EZH2Firstly,we studied whether EZH2 can regulate TGF-β signaling pathway in ATL cell line by reporter gene experiment.It was found that EZH2 had obvious activation and metrological dependence on TGF-β signaling pathway,and the activation effect was confirmed under the condition of reporter gene plasmids using different TGF-βsignaling pathways.And this activation must depend on the existence of Smad3.Subsequently,we constructed a ATL-T stable strain silencing EZH2 by lentivirus infection,and detected the activation of TGF-β signaling pathway in the stable strain by reporter gene experiment.The results showed that the activation of TGF-β signaling pathway was down-regulated in ATL-T stable strains silencing EZH2 compared with the control group.Therefore,this experiment further proves that TGF-β signaling pathway is positively regulated by EZH2 in ATL-T cells.Subsequently,we found that EZH2 protein can bind to Smad3 protein and co-locate in the nucleus.It was found that both EZH2 wild type and mutant without SET domain could activate TGF-β signaling pathway,indicating that EZH2 did not play its histone methyltransferase activity when EZH2 activated TGF-β signaling pathway.Finally,we analyzed the domain of Smad3 protein by immunoprecipitation assay.the results showed that the Linker part of Smad3 protein interfered with the binding of EZH2 protein.Part Ⅲ : The Molecular mechanism of EZH2 regulating TGF-β signaling pathway in ATL cellsIn order to study the molecular mechanism of EZH2 regulating TGF-β signaling pathway,we first used double fluorescein plum reporter gene experiment to find thatEZH2,Smad3 and Smad4 co-activated TGF-β signaling pathway.Then,we proved that EZH2 protein did not affect the stability of Smad3 protein and Smad4 protein by immunoblotting.Subsequently,we found that EZH2 protein can bind to Smad4 protein,which provides a basis for our subsequent experiments.The results of immunoprecipitation test showed that EZH2,Smad3 and Smad4 could form ternary complex,and EZH2 could significantly enhance the binding strength of Smad3 protein and Smad4 protein.Part Ⅳ: Regulation of EZH2 on target gene expression of TGF-βsignaling pathwayIn order to study whether EZH2 can affect the expression of downstream target genes of TGF-β signaling pathway by activating TGF-β signaling pathway,we first used the previously constructed ATL-T stable strain silencing EZH2.Through RT-PCR and qRT-PCR experiments,we found that the expression of ZEB1,ZEB2,Snail1,ADAM9,HDM2 and TWIST1 genes in ATL-T cells silenced with EZH2 was significantly down-regulated in EZH2 silent group.It was proved that the activation of TGF-β / Smad3 signaling pathway by EZH2 was specific in ATL cells.Therefore,we speculate that EZH2 affects the migration and invasion of cells by regulating the expression of target genes of TGF-β signaling pathway,and finally promotes the occurrence and development of ATL.In summary,we found that in ATL cells,HTLV-1 encoded Tax protein can promote the expression of EZH2 protein,which shows high expression in ATL positive cell lines.EZH2 can bind to the Smad3 protein and co-localize in the nucleus,thereby activating the TGF-β signaling pathway,and EZH2 does not exert its histone methyltransferase activity during the regulation.EZH2,Smad3 and Smad4 can form a ternary complex,and EZH2 enhances the binding between Smad3 and Smad4.Synergistic activation of TGF-β signaling pathway ultimately affects the expression of downstream target genes.This topic explains the molecular mechanism by which EZH2 promotes the continuous activation of TGF-β signaling pathway in ATL cells,and provides a new strategy and target for the treatment of ATL with EZH2 as a target.