DNA Methylation Of The MMP-9 Promoter Regulates Collagen Expression In Mouse Cardiac Fibroblasts

Objective:To explore the role of DNA methylation on the regulation of collagen production pathway by MMP-9 in mouse cardiac fibroblasts.Methods:(1)The mouse cardiac fibroblasts(MCFs)of C57BL/6 mice were isolated and cultured by collagenase method.The expression of?-SMA and S100A4 in MCFs was detected by immunofluorescence staining,and the purity of the cells was identified.(2)MCFs were treated with palmitic acid(PA)at 0.01 mmol·L^-1,0.05mmol·L^-1,0.1 mmol·L^-1and 0.2 mmol·L^-1,respectively,for 24 h.Cell viability was detected by CCK-8 method,and m RNA and protein expression of COL1A1 were detected by fluorescence quantitative PCR and Western Blot.(3)After MCFs was treated with 0.05 mmol·L^-1PA for 24 h,the m RNA and protein expressions of COL1A1 and MMP-9 were detected by quantitative fluorescence PCR and Western Blot.(4)To explore the effect of PA on DNA methylation in MCFS:(1)After MCFs was treated with 0.05 mmol·L^-1PA for 24 h,the global methylation level of genomic DNA was measured by ELISA;(2)After MCFs was treated with 0.05 mmol·L^-1PA for24 h,DNA methylation of MMP-9 promoter was detected by Massarray mass spectrometry;(3)After MCFs were treated with 0.05 mmol·L^-1PA for 24 h,the m RNA and protein expressions of DNA methylation transferases(DNMT1,DNMT3A and DNMT3B)and demethylases(TET1,TET2 and TET3)were detected by quantitative fluorescence PCR and Western Blot.(5)MCFs were treated with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine(5-Aza-dc)10?mol·L^-1for 24 h,and then treated with PA 0.05 mmol·L^-1for 24 h.The m RNA and protein expressions of COL1A1 and MMP-9 were detected by quantitative fluorescence PCR and Western Blot.Results:(1)The cells were found to be triangular or spindle-shaped under microscope,and the cell purity was high,which met the requirements of subsequent experiments.(2)Compared with CON group,the cell viability of MCFs was the highest at 0.05mmol·L^-1PA,and the m RNA and protein expressions of COL1A1 were most obviously up-regulated,which was statistically significant compared with CON group(P<0.05).(3)Compared with CON group,m RNA and protein expressions of COL1A1 and MMP-9 in PA group were significantly increased,with statistical significance(P<0.05).(4)PA inhibited DNA methylation levels in MCFs:(1)Compared with CON group,the overall genomic DNA methylation level of PA treated MCFs was significantly down-regulated(P<0.05).(2)Compared with CON group,the methylation levels of two Cp G sites in MCFs MMP-9 promoter sequence after PA treatment were significantly decreased(P<0.05).(3)Compared with the CON group,in PA group the m RNA expression of DNMT3A was down-regulated while the protein expression was up-regulated;the m RNA expression of DNMT3B was up-regulated while the protein expression was down-regulated;the m RNA and protein expressions of DNMT1,TET1,TET2 and TET3 were up-regulated,with statistical significance(P<0.05).(5)MCFs was treated with 5-Aza-dc significantly up-regulated the m RNA and protein expression levels of MMP-9,while the m RNA and protein expression levels of COL1A1 were significantly inhibited(P<0.05).Conclusion:Under the action of PA,the DNA methylation level of MCFs significantly decreased and promoted the expression of MMP-9 and inhibited the produce of collagen,and this result may be related to the expression imbalance of DNA methyltransferase and DNA demmethylation enzyme caused by PA.