| Objective: Atrial fibrillation(AF)is one of the complications of cardiac infarction,hypertension and valvular heart disease.At present,treatment of is atrial fibrillation is difficult and the incidence is on the rise.The mechanism of atrial fibrillation is very complex and it is the focus of cardiovascular research.Atrial fibrillation is mostly associated with Cardiac fibrosis,while the pathogenesis of Cardiac fibrosis is still unclear.Some scholars have conducted many studies and found some valuable conclusions.To elucidate the potential molecular mechanism of the progression of Cardiac fibrosis can provide certain support and assistance for the study and treatment of atrial fibrillation.When the heart is damaged or becomes overloaded,normal cardiac cells are replaced by non-functional fibrotic tissue,leading to varying degrees of cardiac dysfunction,the key is the activation of cardiac fibroblasts.In epigenetics,mechanisms such as DNA methylation,histone modifications,and microrna are involved.Changes in the expression of DNMT3 A,one of the DNA methylation catalytic enzymes,may also play an important role in the progression of Cardiac fibrosis Therefore,it is theoretically possible to correct or inhibit the progression of Cardiac fibrosis by restoring the epigenetic changes.Due to epigenetic changes caused by changes in DNA methylation,MicroRNA expression may have a significant effect on transcription and post-transcriptional gene expression,and thus may be involved in the Cardiac fibrosis process.In recent years,the relationship between microRNA and autophagy has attracted more and more attention.MicroRNAs can be used as biomarkers to monitor and even treat certain diseases by regulating gene expression.Given that autophagy exists in the fibrosis process of cardiac fibroblasts,we can hypothesize that epigenetics prevents cardiac fibrosis by affecting autophagy.MicroRNA is an important regulator of the complex biological process of cardiac fibrosis.However,how it regulates autophagy and fibrosis is still poorly understood.We designed this study to explore the expression of mir-200 b in the autophagy of cardiac cells and its potential function and regulatory mechanism of DNA methylation regulates the expression of microRNA.Recent studies have shown that abnormal DNA methylation in the promoter region of MicroRNA is involved in the pathogenesis of fibrosis.It was found that in fibrotic cardiomyocytes,up to several percentage points of cardiomyocyte-specific DNA were methylated.DNA methyltransferase 3A(DNMT3A)is a member of the methyltransferase family and its promoter specific site methylation leads to silencing of gene expression.The purpose of this study is to explore the regulatory effect of DNMT3 A on miR-200 b in myocardial fibrosis and myofibroblast autophagy.These results may help to develop new effective methods to treat Cardiac fibrosis.Methods: Patients with rheumatic heart disease who underwent heart valve replacement(mitral valve or/and aortic valve replacement)in the cardiac surgery department of our hospital from January 2016 to December 2017 were involved.Patients with hypertension,diabetes mellitus,coronary heart disease,hypertrophic cardiomyopathy and chronic renal insufficiency were excluded.Patients with incomplete clinical data and obvious symptoms of infection were also excluded.According to the presence or absence of combined atrial fibrillation,patients were divided into AF group and SR group.The myocardial tissue specimens were subjected to paraffin pathological sections,immunohistochemical staining,the extraction of RNA from the myocardial tissue,and quantitative qrt-pcr detection of the expression of miR-200 b and Western Blot detection of the relative expression level of DNMT3 A in the isolated myocardial tissue specimens.By replicating the Cardiac fibrosis model of SD rats,40 SD rats were divided into two groups using random number extraction.One group was the experimental group,namely the abdominal aortic stenosis group,and the other group was the blank control group,namely the sham operation group.Abdominal aortic coarctation was performed in rats with abdominal aortic stenosis as described by Hertig,while abdominal aortic coarctation was not performed in sham group.All treatments started 24 hours after surgery and lasted for 4 weeks.Four weeks later,the animals were executed and heart specimens removed.After cell culture and drug treatment,histological analysis was performed,including paraffin pathological sections,HE staining and Masson staining to observe the degree of myofibrosis and collagen volume fraction(CVF)in the heart tissue of rats.The expression levels of miR-200 b,DNMT3A and U6 in the inhibitors and miRNA-200 b mimics and its negative control group were detected by qRT-PCR.The expression levels of DNMT3 A and P62 protein in SD rat heart samples were measured by Western blotting.The effects of inhibitors and microRNA-200 b mimics on the activation and proliferation of cardiac fibroblasts were observed by MTT colorimetry.The number of autophagosomes and the number of autophagic lysosomes were observed by cell transfection and the negative control of small interfering RNA(siRNA)oligomer of DNMT3 A in rats,and the levels of autophagy related genes and protein expression in cells were detected.Autophagy was determined by acridine orange(AO)staining.Results: The expression changes of DNMT3 A and miR-200 b in atrial fibrillation myocardial remodeling: Results of the two groups of SR and AF were compared: positive expression rate of DNMT3 A in the cardiac tissue of the AF group was significantly higher than that of the SR group(P<0.05).Collagen ? in myocardial tissue in the positive expression rate of AF group was obviously higher than that of the SR group(P<0.05).The results of qRT-PCR showed that compared with the AF group,the expression level of miR-200 b in the cardiac remodeling tissues of the AF group was significantly reduced.The difference was statistically significant(p<0.05).Compared with SR group,DNMT3 A mRNA expression in the cardiac remodeling tissues of AF group was significantly increased,and the difference was statistically significant.Compared with the SR group,Col1A1 mRNA expression in the myocardial remodeling tissues of the AF group was significantly increased,and the difference was statistically significant(p<0.05).Western blot results showed that compared with SR group,DNMT3 A protein expression in the cardiac remodeling tissues of AF group was significantly increased,and the difference was statistically significant(p<0.05).Compared with the SR group,Col1A1 protein expression in the myocardial remodeling tissues of the AF group was significantly increased,and the difference was statistically significant(p<0.05).The molecular mechanism of DNMT3 A methylation modified miR-200 b to regulate the autophagy of cardiac fibroblasts: HE staining results showed that collagen fibers in the myocardial tissue stroma of the AAC group were more obvious than those in the Sham group.Compared with Sham group,the AAC group showed disordered myocardial arrangement and myocardial cell hypertrophy.The results of Masson staining showed that the degree of fibrosis was more serious in the AAC group,and the volume fraction of myocardial interstitial collagen(CVF)was significantly increased.The results of qRT-PCR showed that compared with Sham group,the expression level of miR-200 b was significantly decreased in the heart samples,and the difference was statistically significant(p<0.05).Compared with Sham group,DNMT3 A and Col1A1 mRNA expression in AAC group were significantly increased,and the difference was statistically significant.Western blotting confirmed that DNMT3 A protein expression was significantly increased in AAC compared with Sham group,and P62 protein in AAC group was significantly decreased compared with Sham group.Chemical staining further confirmed that compared with the sham operation group,DNMT3 A protein expression was significantly increased in the AAC group,while P62 protein was significantly decreased(p<0.05).qRT-PCR showed that miR-200 b was significantly reduced in the autophagy of cardiac fibroblasts induced by rapamycin,but DNMT3 AmRNA was significantly increased.Western blotting detected the elevation of DNMT3 A protein in the autophagy of myocardial fibroblasts induced by rapamycin.The LC3BII/I ratio was significantly increased after 48 hours of rapamycin treatment,but the expression of P62 was decreased.Acridine orange staining showed that increased red speckle fluorescence was induced in myocardial fibroblasts treated with rapamycin compared with untreated myocardial fibroblasts.Conclusion: DNMT3 A expression was increased in the development of atrial fibrillation myocardial fibrosis,while miR-200 b expression was decreased.Transient transfection of DNMT3A-siRNA resulted in upregulation of miR-200 b expression and inhibition of myocardial fibroblast autophagy.DNMT3 A methylation modified miR-200 b to regulate cardiac fibroblast autophagy plays an important role in the cardiac remodeling of atrial fibrillation.Our results also provide the basis for the molecular mechanism of DNMT3 A and miR-200 b combined with rapamycin to improve the autophagy activity of cardiac fibroblasts.We found that miR-200 b can slow down the process of cardiac fibrosis by inhibiting the autophagy induced by rapamycin.The present work provides theoretical evidence for the molecular mechanism of miR-200 b anti-atrial fibrillation and fibrosis. |
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