| Objective: To study the effect of DNMT1 and MBD1 on the kinetics of DNA methylation on P19 cells Methods:(1)P19 cells were cultured and the lentiviral vector with MBD1 shRNA(#2)was infected into P19 cells,and puromycin was used to screen the infected cells.The knockout efficiency of MBD1 by lentiviral vector with MBD1 shRNA on P19 cells was verified using fluorescence microscopy and qPCR.(2)In order to screen the right concentration of 5-azacytidine(5-Aza),the inhibition rate of 5-Aza on p19 at different concentrations(2.5,5,10,20 and 40μmol/L)and times(24h,48 h,and 72h)were determined by MTT assay,and qPCR and Western blotting were used to determine the optimal time of action of 5-Aza.Meanwhile,MBD1,DNMT1,DNMT3 a,DNMT3b and TET1,TET2,TET3 mRNAs and protein expression levels of P19 cells treated with 5-Aza were detected by qPCR and Western blotting,and 5-mC and 5-hmC protein content were detected by ELISA.(3)P19 cells with #2 lentivirus,MBD1,DNMT1,DNMT3 a,DNMT3b and TET1,TET2,TET3 mRNAs and protein expression levels were detected by qPCR and Western blotting,and 5-mC and 5-hmC protein content were detected by ELISA.Results:(1)The results shown more than 90% P19 cells were expressed GFP after 72 hours infected with lentivirus.qPCR results showed,compared with control group,no difference in MBD1 expression in Scramble group(P>0.05),indicating that the lentivirus itself had no effect on the MBD1 mRNA level;The MBD1 mRNA levels decreased in #2 lentiviruses compared with the Scramble group(P<0.01).Meanwhile,the knockdown effect of #2 lentiviruses was more significant with a knockdown rate of 86.07%.Western blotting results suggested that the MBD1 protein expression of P19 cells decreased in #2 lentiviruses compare with the Scramble group(P<0.01),and the inhibitory rate on the MBD1 protein expression in P19 cells was 68.60%.(2)MTT assay showed that P19 cell treated with 10,20 and 40μmol/L 5-Aza for 24 h,48h and 72 h growth inhibition was thus induced in a dose-dependent manner by 5-Aza.P19 cell treated with 10,20 and 40μmol/L 5-Aza for 24 h,48h and 72 h,DNMT1,DNMT3 a,DNMT3b and 5-hmC mRNAs were detected by qPCR.The results demonstrated that DNMT1 mRNA level at each tested time point was lower in the 5-Aza-treated group compared with that in the control group except for 10μmol/L at 24h(P<0.01),and it decreased following treatment with 5-Aza at a concentration of 40μmol/L for 24 h,48h and 72h(P<0.01);DNMT3b mRNA level significantly increased following treatment with 5-Aza at a concentration of 10μmol/L for 24 h,48h and 72h(P<0.05,P<0.01,P<0.01,respectively),it increased following treatment with 5-Aza at a concentration of 40μmol/L for 48 h and 72h(P<0.01,P<0.01,P<0.01,P<0.01,respectively);5-hmC mRNA levels gradually increased with time and concentration increase(P<0.05,P<0.05,P<0.01,P<0.01,P<0.05,P<0.01,P<0.01,P<0.01,P<0.01,respectively).P19 cells with 20μmol/L 5-Aza,MBD1,TET1,TET2 and TET3 mRNAs levels were detected by qPCR and MBD1,DNMT1,DNMT3 a,DNMT3b TET1,TET2 and TET3 protein expression levels were detected by Western blotting.qPCR results showed that MBD1 and TET1 mRNA levels were increased at 48h(P>0.05)and 72h(P<0.01);TET2 mRNA level increased following treatment with 5-Aza for 24 h,48h and 72h(P<0.01).Western blotting results showed that the protein expression level of MBD1 was increased at 48h(P<0.01);the protein expression level of DNMT1 was decreased at 48 h and 72h(P<0.01);the protein expression levels of DNMT3 a,DNMT3b and TET3 were not changed at all time(P>0.05);the protein expression level of TET1 increased at 72h(P<0.05);the protein expression level of TET2 increased at 24 h,48h and 72h(P<0.05,P<0.01,P<0.01,respectively).5-mC and 5-hmC expression was assessed by ELISA after P19 were treated with 10μmol/L,20μmol/L and 40μmol/L 5-Aza for 24 hours,48 hours and 72 hours.5-mC expression at each tested time point was lower in the 5-Aza-treated group compared with that in the control group except for 10μmol/L at 24h(P<0.05,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,respectively).5-hmC expression at each tested time point was upper in the 5-Aza-treated group compared with that in the control group except for 10μmol/L at 24 h,10 and 20μmol/L at 48h(P<0.01,P<0.01,P<0.05,P<0.01,P<0.01,P<0.01,respectively),and was dose-and time-dependent.(3)P19 cells with #2 lentivirus,MBD1,DNMT1,DNMT3 a,DNMT3b and TET1,TET2,TET3 mRNAs and protein expression levels were detected by qPCR and Western blotting.The results shown that the mRNA levels of DNMT1,DNMT3 a,DNMT3b TET1,TET2,TET3 and 5-hmC were not significantly different between the Scramble group and the control group by qPCR;Compared with the Scramble group,TET1 mRNA levels was not significantly different(P>0.05),and DNMT1,DNMT3 a,DNMT3b,TET2,TET3 and 5-hmC mRNA levels were increased(P<0.05,P<0.05,P<0.01,P<0.01,P<0.01,P<0.05,respectively)in the shMBD1 group;Western blotting showed that there was no significant difference in protein expression levels of DNMT1,DNMT3 a,DNMT3b TET1,TET2 and TET3 between the Scramble group and the control group(P>0.05);Compared with the Scramble group,DNMT1,DNMT3 a,DNMT3b TET1 and TET3 protein expression levels were increased(P<0.01,P<0.01,P<0.01,P<0.05,P<0.05,respectively)in the shMBD1 group,but no significant change in expression level of TET1 protein(P>0.05).The ELISA results showed that there was no significant difference in expression levels of 5-mC and 5-hmC between the Scramble group and the control group(P>0.05);Compared with the Scramble group,5-mC expression levels was decreased(P<0.01)in the shMBD1 group,but increased in expression level of 5-hmC(P>0.05),and was dose-and time-dependent.Conclusion: In this study,the resuts shown 5-Aza significantly reduced DNMT1 mRNA level and protein expression level,and MBD1 shRNA significantly decreased the expression level of MBD1 mRNA and protein expression level.And our research data suggested MBD1 and DNMT1 interact with each other to maintain the dynamic balance of DNA methylation.DNMT1 interacts with TET3,however,MBD1 maintains the homeostasis of 5-mc and 5-hmc in P19 cells by directly regulating TET1.Our results shown the DNMT1 and MBD1 play an important role in maintaining the homeostasis of DNA methylation kinetics. |
PDF Full Text Request