DNA Methylation Regulates ?-smooth Muscle Actin Expression During Cardiac Fibroblast Differentiation

Part I: Cardiac fibrosis post-myocardial infarction is associated with DNA methylationObjective: To explore the relationship between cardiac fibrosis and DNA methylation after myocardial infarction(MI).Methods: Sprague Dawley rats were randomly divided into MI group and Sham group.Six weeks after operation,the area of fibrosis was determined by H&E staining and Masson staining.The expression of fibrosis-related factors was detected by q PCR,ELISA and immunofluorescence.Immunohistochemistry was used to detect the proliferation in cardiac fibrosis tissues.DNA methyltransferases activity assay kit was used to analysis the activity of DNMTs.QPCR and western blot were used to detect expression of ?-smooth muscle actin(?-SMA)and DNA methyltransferases(DNMTs)in fibrotic tissues.Results: Compared with the Sham group,the expression of procollagen I C-terminal Propeptide(PICP)in the peripheral blood of the MI group and the fibrosis-related molecules in the corresponding tissues were significantly upregulated.The activity of DNMTs was significantly inhibited,and the expression of DNMT1 was significantly downregulated in the corresponding tissues of MI group.Conclusion: Cardiac fibrosis post MI is associated with DNA methylation.Part II: DNA methylation is involved in the differentiation and proliferation of cardiac fibroblastsObjective: To investigate the regulation mechanism of DNA methylation in the differentiation and proliferation of cardiac fibroblasts(CFs)induced by transforming growth factor ?1(TGF-?1).Methods: Primary CFs were isolated and cultured and identified by immunofluorescence.CFs was treated with TGF-?1 to induce differentiation.To explore whether ?-SMA expression was regulated by DNMT1,CFs were transfected with pc DNA-DNMT1 or si DNMT1,and the expression of ?-SMA and DNMTs was detected by q PCR and western blot.Bisulfite sequencing analysis was used to explore the DNA methylation level in the promoter region of ?-SMA gene.CCK-8 and Ed U incorporation assay were used to explore the proliferation of CFs.Results:(1)Compared with the control group,the expression of ?-SMA in CFs was increased after TGF-?1 treatment,while the expression of DNMT1 was significantly downregulated.(2)DNMT1 regulated the expression of ?-SMA in CFs,and overexpression of DNMT1 significantly abrogated the TGF-?1-induced increase in ?-SMA expression;while knockdown of DNMT1 supported the TGF-?1-induced increase in ?-SMA expression.(3)Compared with the control group(37.5%),the methylation level of Cp G island in the ?-SMA promoter region after TGF-?1 treatment was significantly decreased(12.5%),while overexpression of DNMT1 prevented TGF-?1-induced demethylation of the ?-SMA promoter(33.3%).(4)TGF-?1 mediates cardiac fibroblast proliferation by regulating DNMT1.Conclusion: Taken together,we demonstrated that ?-SMA expression during CF differentiation was regulated by demethylation of its gene sequence through the inhibition of DNMT1 expression.These results indicate the importance of DNA demethylation and DNMT1 in the induction of myofibroblast differentiation and reveal DNMT1 as an additional potential target for controlling CF differentiation and cardiac fibrosis.Part III TGF-?1 regulates proliferation and differentiation of cardiac fibroblasts via Smad and MAPK signaling pathwaysObjective: To study whether Smad or MAPK signaling pathways are involved in TGF-?1-induced CF differentiation and proliferation.Methods: CCK-8 and Ed U incorporation assay were used to explore the proliferation of CFs and western blot was used to detect the protein levels of ?-SMA,DNMT1,Smad 2/3 and the three well-characterized MAPK subfamilies,JNK,p38,and ERK,as well as their phosphorylated forms in CFs with TGF-?1 alone or coupled with pSmad2/3 inhibitor SB431542,p-JNK inhibitor SP100625,p-p38 inhibitor SB203580,or p-ERK inhibitor AZD6244.Results:(1)The western blot results demonstrated increased protein expression levels of p-Smad 2/3,p-JNK,p-p38,and p-ERK,but without alterations in Smad 2/3,JNK,p38,and ERK protein expression levels in CFs treated with 10 ng/ml TGF-?1.Meanwhile,activation of relative phosphorylated forms was repressed by their corresponding inhibitors.(2)The CCK-8 assay results demonstrated that treatment with the inhibitors all could restore the TGF-?1-induced cell proliferation compare to TGF-?1 group.And the results of Ed U assay is consistent with CCK-8 assay.Conclusion: TGF-?1 promote the proliferation of CFs through both Smad and MAPK signaling pathway and the Smad and MAPK signaling pathway are both involved in the regulation of DNMT1 and ?-SMA expression induced by TGF-?1.