Mechanism Of DNMT1 Methylated MiR-152-3p Regulating Activation And Proliferation In The Cardiac Fibroblast

Objective The occurrence of atrial fibrillation is closely related to the progress of myocardial fibrosis.Activated Cardiac fibroblasts(CFs)turn into myofibroblasts and secrete extracellular matrix(ECM)proteins is the key to the development of myocardial fibrosis,but the specific mechanism is not completely clear.In recent years,a large number of studies have shown that epigenetics plays an important role in the occurrence and development of cardiac fibrosis while microRNA and DNA methylation are the key objects.Micro RNA-152-3p is a member of microRNA.Studies have found that the expression of microRNA-152-3p in tumors is inversely related to DNA methylation-modified which has been reported in liver fibrosis and lung fibrosis.DNA methylation has been shown to be involved in the development of cardiac fibrosis.This study preliminary investigated the role of DNMT1 and microRNA-152-3p in the process of myocardial fibrosis by studying the relationship between DNMT1(DNA methyltransferase 1),microRNA-152-3p and myocardial fibroblast activated proliferation while the possible relationship between DNMT1 and microRNA-152-3p in myocardial fibrosis.Methods Forty Sprague-Dawley(SD)male rats were purchased for experiments.All of them were 8-week-old age,healthy and weighed about 200±20 g.The rats were randomly divided into two groups.Among them,there were 20 in the ISO group(subcutaneous injection model group of Isoproterenol 5mg/kg/d)and 20 in the normal saline group(subcutaneous injection group of equal normal saline).The heart was removed after 2 weeks of continuous injections.The pathological methods of HE,Sirius Red and Masson staining were used to detect whether the heart of the rats in the ISO group had fibrotic lesions.Cardiac myofibroblasts(CFs)were extracted and cultured From SD neonatal rat.After activation of TGF-?1 for 24-48 hours,microRNA-152-3p mimics,inhibitors and DNMT1-si RNA(DNMT1 small interference)were transfected into CFs transiently and the corresponding negative control(NC).Cells were treated after 24 to 48 hours.Western blot was used to determine the expression of ?-SMA,Collagen?,and DNMT1 proteins in rat hearts and CFs.qRT-PCR was used to detect the expression of miR-152-3p,DNMT1,Collagen?,and ?-SMA in cardiac tissues and CFs.CCK-8 assay was used to detect the cell viability of the extracted cardiac fibroblasts.Results Compared with the normal saline group,HE,Sirius Red,and Masson staining results showed that there was a large amount of collagen deposition in the interstitial of myocardial cells and the arrangement was disordered in the ISO group;qRT-PCR results showed that the expression of miR-152-3p was significantly reduced The m RNA expression levels of Collagen?,?-SMA and DNMT1 were significantly increased;Western blot results showed that the expression levels of Collagen?,?-SMA and DNMT1 proteins were increased.In vitro experiments,the viability of TGF-?1activated CFs and the expression of Collagen?,?-SMA,DNMT1 were increased,but the expression of miR-152-3p decreased.In comparison of CFs transfected with TGF-?1 stimulation in each group,qRT-PCR showed that the expression of miR-152-3p increased,but Collagen?,?-SMA decreased in mimics group.The expression of miR-152-3p decreased while Collagen? and ?-SMA increased significantly in inhibitors group.Compared with the negative control,qRT-PCR showed that the expression of miR-152-3p increased in DNMT1-si RNA group.Compared with the negative control,western blot results showed that the protein expressions of Collagen?,?-SMA were decreased in the mimics group but increased in inhibitors group;the protein level of Collagen?,?-SMA,and DNMT1 were significantly reduced in DNMT1-si RNA group.CCK-8 assay showed that the cell vabilitiy of CFs was significantly enhanced in inhibitors group but decreased in mimics group and the DNMT1-si RNA group.Conclusion In the ISO-induced SD rat model of cardiac fibrosis,the expression of Collagen ?,?-SMA and DNMT1 increased while the expression of miR-152-3p decreased.In vitro experiments,CFs were treated by TGF-?1,the high expression of miR-152-3p can inhibit the expression of Collagen?,?-SMA while the low expression can promote the expression of Collagen? and ?-SMA as well as the low expression of DNMT1 upregulate the level of miR-152-3p.These results indicate that miR-152-3p may play an important role in the occurrence and development of myocardial fibrosis and is affected by the expression of DNMT1.