Role Of MiR-369-5p Methylation Regulation Of PTCH1 Expression Changes In Atrial Fibrosis

Objective Atrial fibrosis plays an important role in the structural changes of atrial fibrillation.The pathological anatomy of atrial fibrillation is atrial fibrosis and atrial dilatation.The early manifestations of electrical remodeling and ion channel characteristics change.The course of the disease progressed,myocardial cell hypertrophy,atrial fibrosis,collagen deposition and other structures changed.During myocardial fibrosis,cardiac fibroblasts are affected by some signaling factors.In recent years,miRNAs have been widely involved in the regulation of cardiovascular system function.Among them,miRNAs have been involved in the regulation of atrial remodeling to affect atrial fibrillation.Revealed.Micro RNA-369-5P is a micro RNA.Studies have shown that micro RNA-369-5P can mediate DNA methylation,which in turn changes the expression of PTCH1.DNA methylation has been shown to be involved in atrial fibrosis.Development has taken place.This study will use micro RNA-369-5P as an entry point to detect the expression of downstream target genes and proteins by affecting its expression,and observe its effect on the activation and proliferation of rat cardiac fibroblasts(CFs)to investigate micro RNA-Possible mechanism of action of 369-5P.Methods Fifty male Sprague-Dawley rats aged 6 weeks and with similar body weight were randomly divided into 2 groups,which were labeled as AAC group(a model of myocardial fibrosis induced by abdominal aortic coarctation),and sham operation group.Twenty rats(Sham technique)were sacrificed 4 weeks after surgery to obtain cardiac specimens.HE staining and Masson staining were used to observe the pathological changes of rat cardiac specimens and calculate the collagen volume fraction(CVF).SD rat neonatal rat cardiac fibroblasts(CFs)were cultured,and micro RNA-369-5p mimics and inhibitor and its negative control and DNMT3 a overexpression plasmid group were transiently transfected into SD neonatal rat cardiac fibroblasts with Lipofectamine TM 2000 Reagent reagent.(m DNMT3a-p EGFP-C3),small interfering DNMT3 a si RNA group and negative control group,treated cells after 24~48h,detected the expression of miR-369-5p by q RT-PCR,and DNMT3 a,PTCH1,type I collagen and ?-smooth muscle The m RNA level of actin(?-SMA)was expressed,the expression of ?-SMA,Col I,PTCH1 and DNMT3 a protein was detected by Western blot,and the proliferation activity of extracted cardiac fibroblasts was detected by CCK8.Results Compared with the sham operation group(Sham technique),HE and Masson staining showed that the collagen fibers in the interstitial tissue of the heart tissue increased,the collagen volume fraction(CVF)increased,and the fibrosis model succeeded;q RT-PCR results showed that m RNA expression levels of miR-369-5p and PTCH1 were significantly decreased,and m RNA expression levels of Col I,?-SMA and methyltransferase 3A(DNMT3a)were significantly increased.The results of Western blot showed that the expression of Col I,?-SMA and DNMT3 a increased,and the expression of PTCH1 protein decreased.In cell experiments,q RT-PCR was used to transiently transfect micro RNA-369-5p mimics and inhibitors and their negative control primary CFs,suggesting that miR-369-5p and PTCH1 m RNA expression is up-regulated in mimics CFs,Decreased m RNA expression levels of?-SMA,DNMT3 a and Col I;q RT-PCR was compared with the DNMT3 a si RNA group in the primary CFs transfected with m DNMT3a-p EGFP-C3 and its negative control and DNMT3 a si RNA and its negative control.The m RNA expression levels of Col I,?-SMA and DNMT3 a were significantly increased in the m DNMT3a-p EGFP-C3 group,and the m RNA expression level of PTCH1 was decreased.Western blot results showed that the expression of Col I,?-SMA and DNMT3 a was decreased and the protein expression of PTCH1 was increased in the mimics group.The expression of protein of Col I,?-SMA and DNMT3 a was increased in the m DNMT3a-p EGFP-C3 group,and the protein expression of PTCH1 was decreased..The results of CCK8 experiments showed that the proliferation activity of the cells was significantly enhanced in the m DNMT3a-p EGFP-C3 and inhibitors groups.Conclusion The expression of miR-369-5p is decreased in the myocardial fibrosis model of AAC,the expression of Col I,?-SMA and DNMT3 a is increased,and the expression of PTCH1 is decreased.In cell experiments,high expression of miR-369-5p inhibited the expression of Col I,?-SMA and DNMT3 a,while decreased expression of DNMT3 a up-regulated the expression of PTCH1.These results suggest that miR-369-5p mimics can inhibit the development of myocardial fibrosis and is related to the expression of genes involved in methylation inhibition.PTCH1 may be a potential target of miR-369-5p.