The Regulatory Mechanism Of MicroRNA-200a In Cardiac Fibrosis And The Effect Of Plasma Large Endothelin-1 On The Prognosis Of Patients With Atrial Fibrillation

Part 1:MicroRNA-200a suppresses the activation and pro-fibrogenic effects of cardiac fibroblasts by targeting endothelin receptor type A and transforming growth factor-? receptor type 2Objective:Atrial fibrillation is the most common arrhythmia encountered in the clinical practice,and it significantly increases the morbidity and mortality,leading to a great social and economic burden.The pathophysiological mechanism of atrial fibrillation is intricate,and atrial structural remodeling characterized by fibrosis plays a vital role in promoting the formation of complex reentry mechanism and the initation,development,and maintenance of atrial fibrillation.MicroRNAs,as a class of small non-coding RNAs,are inloved in a series of life processes and pathophysiological processes,and an increasing body of evidence have established that microRNAs play an important regulatory role in the development of atrial fibrillation.Recent studies have indicated that microRNA-200a is involved in the process of fibrotic remodeling,however,the specific mechanism of microRNA-200a in cardiac fibroiss is unclear The objective of the present study was to investigate the regulatory role of microRNA-200a in cardiac fibrosis via in vitro experiments,which prodived insights in the regulatory mechanisms of microRNA-200a in the initiation,development,and maintainence of atrial fibrillation.Methods:Transforming growth factor-? type 2(Tgfbr2)and endothelin receptor type A(Ednra)were identified to be target genes of microRNA-200a by microRNA target gene prediction websites.Angiotensin II(ANGII)was added to isolated and cultured primary mouse cardiac fibroblasts to construct fibrosis model,and the expression of microRNA-200a was detected by quantitative real time polymerase chain reaction(qRT-PCR).MicroRNA,200a mimics and microRNA-200a inhibitors were transferred into cells using LipofectamineTM RNAiMAX,and the expression of a-smooth muscle action(a-SMA),type ? collagen,type ? collagen,TGFBR2,and EDNRA were examined by qRT-PCR and western blot.Cell proliferation after transfection of microRNA-200a mimics and inhibitors was dected using Cell counting kit-8.The dual luciferase reporter assasy was used to verify the direct binding of microRNA,200a to Tgfbr2 and Ednra.Results:The expression level of microRNA-200a was significantly reduced in the cell fibrosis model constructed by ANGII-stimultated mouse cardiac fibroblasts.After transferring microRNA-200a mimics into mouse cardiac fibroblasts,the expression of TGFBR2 and EDNRA was significantly decreased,as well as the expression of a-SMA,type ? collagen,and type ? collagen,besides,cell proliferation was significantly lower than that of the mimic negative control group.In contrast,the expression of TGFBR2 and EDNRA in mouse cardiac fibroblasts was significantly increased after microRNA-200a inhibitors were transferred into cells,as well as the expression of a-SMA,type I collagen,and type ? collagen,besides,cell proliferation was significantly higher than that of the inhibitor negative control group.The dual luciferase reporter assay further verified that Tgfbr2 and Ednra were direct target genes of microRNA-200a.Conclusion:The expression level of microRNA-200a is significantly down-regulated in ANGII-stimulated mouse cardiac fibroblasts,and the down-regulated expression of microRNA-200a promotes the activiation,proliferation,and collagen production of mouse cardiac fibroblasts by reducing the inhibitory effects on its downstream target genes of Tgfbr2 and Ednra,which further aggravates the fibrosis process.microRNA-200a attenuates the proliferation,activation and collagen production of mouse cardiac fibroblasts by inhibiting Tfbr2 and Ednra,suggesting that microRNA-200a might have potential therapeutic value in preventing cardiac fibrosis.Part 2:The impact of plasma big endothelin-1 levels at admission on long-term outcomes in patients with atrial fibrillationObjective:Atrial fibrillation is a common tachyarrhythmia encountered in clinical practice,which leads to the deterioration of cardiac function caused by tachycardia and the increased risk of thromboembolism,and it is necessary to establish new risk factors as to facilitate the prognostic assessment and risk stratification in patients with atrial fibrillation.As an important component of the fibrosis signaling pathway network,endothelin-1(ET-1)plays a vital role in the negative remodeling of atrial fibrillation.Existing studies have revealed that elevated ET-1 levels are independent risk factors for the incidence of atrial fibrillation,however,scarce studies have assessed the relationship between ET-1 levels and long-term prognosis in patients with atrial fibrillation.The present study aimed to evaluate the prognostic value of ET-1 levels in patients with atrial fibrillation.Methods:The present study was designed as a single-center based,retrospective observational study and aimed to evaluate the relationship between plasma big ET-1 levels and long-term prognosis in patients with atrial fibrillation.A total of 716 patients with a definite diagnosis of atrial fibirllation were consecutively recruited from 2009 to 2015.Patients' baseline information was collected and recorded by reviewing clinical records and electronic databases.The concentrations of big ET-1 were measured using big ET-1 ELISA Kit(BI-2008 2H,Biomedica,Wien,Austria).Follow-up were conducted via clinic visits,telephone,or delivery of medical records by trained research personnel,and completed in August 2017,with a median follow-up duration of 3 years.The primary outcomes were all-cause mortality and combined endpoint events(CEEs).CEEs were defined as a composite of all-cause mortality,non-fatal stroke,non-central nervous system(CNS)embolism,and major bleeding.The secondary outcomes were cardiovascular death,thromboembolic events,and major bleeding.P articipants were divided into high big ET-1 levels group and low big ET-1 levels group,based on the optimal cut-off value of big ET-1 in predicting all-cause death,which was determined by performing receiver-operating characteristic(ROC)curve analysis.Between-group comparisons were performed using Pearson's Chi-squared test or Fisher's exact test for categorical variables and the paired Student's t-test or the Mann-Whitney U test for continuous variables.Logistic regression analysis was used to identify factors related to high big ET-1 levels.Survival curves were depicted using the Kaplan-Meier product-limit method,and comparisons were performed using Log-rank tests.Cox proportional hazards regression models were constructed to test the associations between variables and outcomes.The incremental contribution of big ET-1 in predicting the risk of all-cause mortality and CEEs was evaluated using net reclassification improvement(NRI)approach and integrated discrimination improvement(IDI)analysis.Results:The mean age of the study population was 63.7 � 12.6 years,and 327 patients(45.7%)were female.The C statistic of plasma big ET-1 to predict all-cause death was 0.772(P<0.001),and the best cut-off value was 0.55 pmol/L.Compared to patients with big ET-1<0.55 pmol/L,patients with big ET-1>0.55 pmol/L had notably more comorbidities and worse cardiac functions.Logistic regression analysis revealed that higher NT-pro-BNP values,lower left ventricular ejection fraction values,and larger left atrial diameter were factors associated with big ET-1>0.55 pmol/L.During a median follow-up of 3 years,191 deaths(7.8 per 100 person-years)and 237 CEEs(9.8 per 100 person-years)occurred;143 patients(5.8 per 100 person-years)underwent cardiovascular deaths,66 patients(2.8 per 100 person-years)had thromboembolic events,and 21 patients(0.9 per 100 person-years)experienced major bleeding.Compared to the big ET-1<0.55 pmol/L group,patients with big ET-1>0.55 pmol/L had notably a higher incidence of all-cause mortality(12.5 vs.3.5 per 100 person-years,P<0.001),CEEs(14.8 vs.5.3 per 100 person-years,P<0.001),cardiovascular death(9.7 vs.2.3 per 100 person-years,P<0.001),major bleeding(1.3 vs.0.5 per 100 person-years,P=0.016),and tended to have increased thromboembolic risk(3.4 vs.2.4 per 100 person-years,P=0.071).After multivariate adjustment,big ET-1>0.55 pmol/L was still a predictor of all-cause death(harzard ratio[HR]2.03,95%confidence interval[CI]1.42-2.92;P<0.001),CEEs(HR 1.73,95%CI 1.27-2.36;P=0.001),and cardiovascular death(HR 2.32,95%CI 1.48-3.64;P<0.001).NRI analysis indicated that the addition of big ET-1>0.55 pmol/L to the models constructed by other identified predictors provided an improvement of 0.32 in risk prediction for both all-cause death(95%CI 0.18-0.40;P<0.001)and CEEs(95%CI 0.16-0.40;P=0.027),and IDI analysis indicated that the addition of big ET-1>0.55 pmol/L provided an increase of 0.018 for all-cause death(95%CI 0.001-0.041,P=0.013)and 0.012 for CEEs(95%CI 0.000-0.031,P=0.033).Conclusion:Elevated big ET-1 levels is independently associated with long-term all-cause mortality,CEEs,and cardiovascular death in patients with AF,and further NRI and IDI analysis indicate that big ET-1>0.55 pmol/L can provide incremental contribution in predicting all-cause mortality and CEEs,suggesting that plasma big ET-1 might be valuable for predicting poor prognosis in patients with atrial fibrillation.