Roles Of Polycomb Family Interaction With Gata-6 And PPAR? In Cardiac Differentiation Of P19CL6 Cells

BackgroundWith the development of science and technology and the improvement of human living standards,the incidence of cardiovascular diseases has been increasing year by year,and myocardial infarction has become one of the main causes of death in the world.Although many measures have been successfully applied to treat myocardial infarction,these techniques cannot fundamentally repair the damaged myocardial cells.In recent years,the use of stem cell transplantation to treat myocardial infarction has attracted the attention of scientists.In particular,stem cells(SCs)as a renewable resource of human cardiac myocytes,have broad development prospects.P19CL6 cells are mouse teratoma cells.Under the treatment of 1% DMSO,P19CL6 cells can be directed to differentiate into myocardial cells that can beat rhythmically.In this study,this model was used to study myocardial differentiation in vitro.As one of the members of the nuclear receptor family,Peroxisome proliferatoractivated receptor alpha(PPAR alpha)is a molecular target of bate lipid-lowering drugs,which can regulate the intake,esterification and oxidation of fatty acids by regulating the expression of key genes in energy metabolism.GATA family is a kind of transcription factors with zinc finger structure,in which Gata-6 is responsible for regulating embryonic morphogenesis and tissue specific differentiation,and has been recognized as an essential gene for heart development.Studies have shown that Gata-6 recruits PPAR? to the glucose transporter Glut4,which regulates myocardial energy metabolism.As we all know,myocardial differentiation is closely related to energy metabolism.So,how do Gata-6 and PPAR? affect the differentiation process of P19CL6 cells?Therefore,we started with the ligand finofibrate of PPAR? and the antagonist GW6471 to preliminarily explore the changes in the differentiation process of P19CL6 cells,so as to further study the effect of PPAR? on the formation of myocardial cells.How about the effect of Gata-6,which can form a complex with PPAR?,on the differentiation of myocardium? According to previous research results,Gata-6 could obviously promote the P19CL6 cells to myocardial cell differentiation,while PPAR? in P19CL6 cells had no obvious effect on differentiation after expression.On the contrary,the total collaborative role of PPAR? and Gata-6 in the P19CL6 cells differentiation was not obvious,and even inhibited P19CL6 cells to myocardial cell differentiation.Interestingly,transient transfection of PPAR alpha and/or Gata-6 had a significant synergistic activation of myocardial marker gene alpha-MHC promoter activity.In order to explain this phenomenon,we speculated whether other auxiliary inhibitory factors were recruited during the interaction between PPAR alpha and Gata-6,thus affecting the promotion of PPAR alpha and Gata-6 on P19CL6 differentiation into myocardium.Given that Polycomb family proteins(PcG)are a group of proteins that inhibit gene transcription,we wondered whether Polycomb was involved in this process.Literature search revealed that the 299 lysine of Gata-4 protein can be methylated by PcG member EZH2 to inhibit its transcriptional activity.In addition,Gata-4 also affects the proliferation and differentiation of tumor cells by recruiting polycomb family proteins.Therefore,based on the structural and functional similarities among the protein molecules of the same family members,we conjectured that PcG might interact with PPAR alpha and Gata-6 to inhibit P19CL6 cell differentiation into myocardium.From the above,we launched the research of this subject.PurposeThis study aims to explore the effect of the interaction of Polycomb family with Gata-6 and PPAR? on the differentiation of P19CL6 cells,which will be of great significance for the study of stem cell transplantation and the treatment of myocardial infarction.Methods 1.Two or six days during the induction of P19CL6 cells,different concentrations of PPAR alpha agonist phenotetrol,antagonist GW6471 or deoxyepinephrine were used to collect nucleoprotein and RNA for western blotting and q PCR to detect the expression of myocardial marker gene alpha-MHC.2.Overexpression or inhibition of PPAR alpha and Gata-6 expression in P19CL6 cells,western blotting was used to detect whether the number of days when P19CL6 cells differentiated into cardiomyocytes was affected.3.Different domains of PPAR alpha and Gata-6 were transfected into P19CL6 cells to investigate the effect of PPAR alpha and Gata-6 interaction on myocardial differentiation.4.Pc G,PPAR? and Gata-6 were transfected with luciferase reporter plasmid containing ?-MHC promoter into P19CL6 cells.Cells were collected 36 h later to detect the luciferase activity.5.P19CL6 cells were differentiated into cardiomyocytes by 1% DMSO,and the expressions of EZH2,BMI1,Ring1 B,PPAR?,and Gata-6 were detected by western blotting.6.PPAR? and Gata-6 were transfected into P19CL6 cells with EZH2,Ri-EZH2,BMI1 and Ri-BMI1 respectively or together,with liposome transfection method,and G418 was used to sift cells for 2 weeks to obtain cell lines with stable expression of the corresponding genes,and western blotting was performed to verify the expression of the above genes.Then the stable transfected cell lines were induced to differentiate into cardiomyocytes using DMSO,and the cells were collected to extracte nuclear proteins,and the expression of ?-MHC protein was detected by western blotting.7.Undifferentiated and differentiated P19CL6 cells were collected to perform Ch IP experiments.The corresponding proteins were IP by anti-Pc G,anti-PPAR?,antiGata-6 and antibodies related epigenetic modification markers,and DNA fragments were obtained by purification column.To detect the enrichment of these proteins on ?-MHC promoter,we conducted q PCR experiments.8.Differentiated P19CL6 cells were collected.Nucleoprotein was extracted to conduct Co-IP experiment.Antibodies of PPAR? and Gata-6 were added,and the pulled proteins were used for western blotting to detect whether EZH2,Bmi1 and Ring1 B interacted with them.Results 1.The induced differentiation results of P19CL6 cells treated with the drug were as follows:(1)Agonist of PPAR alpha fenofibrate affects P19CL6 cell differentiation process,in a dose and time dependent manner.With the increase of fenofibrate dose and duration of the action of extended,myocardial differentiation marker gene alpha-MHC protein induced by the time extended to 12 days after 18 days,and the combination of fenofibrate and Phe in P19CL6 cells,inhibits the differentiation process.(2)the PPAR alpha antagonist GW6471 affects P19CL6 cell differentiation process also in a dose and time dependent manner,but in contrast to the fenofibrate trend,with the increase of the dose and duration of the action of extended,alpha-MHC proteins induced by the time 16 days to 12 days ahead of schedule.Coordination with GW6471,Phe can promote P19CL6 cell differentiation.2.Overexpression of PPAR alpha has no effect on P19CL6 cell differentiation,but inhibition of PPAR alpha prolonges the time of myocardial cell formation.Overexpression of Gata-6 can advance the occurrence time of alpha-MHC protein to the 14 th day of induction,while after inhibiting the expression of Gata-6,no expression of alpha-MHC was observed in P19CL6 cells for 18 days.3.The synergistic effect of Gata-6 and PPAR alpha inhibits the differentiation of P19CL6 cells,but the interactions of different domains of Gata-6 and PPAR alpha produce different effects on the differentiation of P19CL6 cells,some promoting,some inhibiting,indicating that the mechanism of Gata-6 and PPAR alpha on the differentiation may be related to their domains.4.The experimental results of luciferase for detecting the activity of the alpha-MHC promoter are as follows:(1)In P19CL6 cells,transfected with Gata-6 or PPAR alpha alone,the alpha-MHC promoter was significantly activated,while co-transfected Gata-6 and PPAR alpha,the activity of alpha-MHC promoter was activated more than 10 times compared with the control group,and significantly higher than in the separate transfection group.(2)In the co-transfection group of Pc G,Gata-6 and PPAR alpha,the alpha-MHC promoter was not significantly activated compared with the control group,indicating that Pc G could inhibit the activation of Gata-6 and PPAR alpha on the alpha-MHC promoter.5.The differentiation of P19CL6 cells was induced,and the expression of alpha-MHC protein was expressed at day 12.During the differentiated process,PPAR alpha and Gata-6 proteins were always expressed,EZH2 and BMI1 were increased with the differentiation process,while Ring1 B were decreased with the differentiation.The dynamic changes of Pc G proteins expression suggest that it may play a specific regulatory role in the process of myocardial differentiation.6.Twelve cell lines of PPAR alpha,Gata-6,EZH2,Ri-EZH2,Bmi1 and Ri-Bmi1 were stabilized and induced to differentiate into myocardium.Among them,only Gata-6+Ri-EZH2 and Gata-6+PPAR alpha +Ri-EZH2 cell lines showed the expression of alpha-MHC on day 16,while no other cell lines showed the expression of alpha-MHC,suggesting that Pc G overexpression or knockdown had an effect on the differentiation of P19CL6 cells.7.Chip-q PCR results showed that in addition to PPAR alpha,gata-6,EZH2,BMI1,Ring1 B,H3K9me3,H3K27me3,HP1 alpha,HP1 beta,and HP1 gama were significantly enriched at the alpha-MHC promoter compared with that before differentiation.8.Co-IP results showed that Pc G proteins could bind to both PPAR alpha and Gata-6.These results suggestted that EZH2,Bmi1,and Ring1 B could regulate P19CL6 cell differentiation by binding to PPAR alpha and Gata-6.Conclusions 1.EZH2,Bmi1 and Ring1 B acted on P19CL6 cells alone or together,and all of them inhibited the activation of Gata-6 and PPAR alpha on alpha-MHC promoter.2.Pc G overexpression or knockdown inhibited the differentiation of Gata-6,PPAR alpha and Gata-6+PPAR alpha.In particular,the inhibitory effect of Pc G overexpression on the differentiation of P19CL6 cells into cardiomyocytes was more obvious.3.Pc G,H3K9me3 and H3K27me3 were enriched on the alpha-MHC promoter,and Pc G formed a protein complex by binding Gata-6 and PPAR alpha,which ultimately inhibiting P19CL6 differentiation into cardiac myocytes.