Expression Of MicroRNA In Arrhythmogenic Right Ventricular Cardiomyopathy And Its Regulatory Mechanism

IntroductionArrhythmogenic right ventricular cardiomyopathy (ARVC), a typically autosomal dominant heart muscle disease coupled with ventricular enlargement, dysfunction and lethal arrhythmias, is the primary reason of sudden death in young people and athletes. The estimated prevalence of ARVC in the general population is about 1/2000-5000. The hallmark pathogenesis of ARVC is the myocardium tissue being replaced by fibro-fatty. During the last two decades, the genetic analysis of ARVC fostered the view that it is a desmosomal dysfunction disease. Until now several causative desmosomal mutations of genes have been discovered in ARVC including, plakoglobin (PG), desmoplakin (DSP), plakophilin-2 (PKP2), desmoglein-2 (DSG2) and desmocollin-2 (DSC2). Moreover, extra rare extra desmosomal genes have also been identified, such as the transforming growth factor-P3 gene (TGF-β3) and connexin43 (Cx43).Wnt/p-catenin pathway is well known for its pathogenic roles as a key regulator of myogenesis versus adipogenesis. Wnt/β-catenin signaling is a key regulator of myogenesis versus adipogenesis. Previous studies showed through suppressing expression of DSP led to the release of PG from the desmosomes. PG could change its location to nucleus and through competition with β-catenin to suppress the canonical Wnt/β-catenin pathway, thereby enhancing adipogenesis induced by PPARy and C/EBP-a. It is recognized that there is a crosstalk between Wnt/β-catenin and Hippo/YAP signaling pathways, the crosstalk formation of protein is complex between YAP, PG, and β-catenin. Molecular remodeling of the intercalated discs leads to pathogenic activation of the Hippo pathway, activation of NF2 was associated with cascade phosphorylation of the Hippo kinases, including MST1/2, LATS1/2, and YAP resulting in increased localization of phosphorylated YAP, suppression of the canonical Wnt signaling, and enhanced adipogenesis in ARVC. The molecular mechanisms of fibrofatty change and regulatory pathways that control ARVC remain largely unknown.Recent studies have manifested that microRNAs play widespread roles in various facets of cardiac function through gene regulation and other cellular processe. Numerous studies indicated that microRNAs have been orchestrated in regulation of cardiac development and function. The Myocardial microRNAs play essential role in complicated biological cardiac disease, including actue myocardial infarction, cardiac hypertrophy, heart failure, atherosclerosis et al. In addition, micro RNAs were associated with various biological processes through cross talk between the canonical Wnt signaling pathway and Hippo signaling pathway. However, the connection between ARVC and microRNA(s) is still largely unknown.Objective:This study focuses on exploring the relationship between microRNA and ARVC, by using technologies of molecular biology, molecular genetics, and qRT-PCR. The study aimed to delineate the molecular pathogenesis of ARVC, to investigate the microRNA function of adipogenesis on Hippo pathway to regulate target gens in ARVC.Methods and resultsBy using a sensitive S-Poly(T) microRNA real-time polymerase chain reaction (RT-PCR) technology, the expression of 1078 human microRNAs were profiled in RNA pools obtained from either right ventricular tissue of 24 heart transplantation ARVC patients or that of 24 healthy hearts, which led to the identification of 24 significantly altered microRNAs candidates. Further analysis in each ARVC tissue validated that 12 of these microRNAs were upregulated and 11 microRNAs were down regulated.Combining the lines of evidence from relative ROC analysis, all the validated microRNAs were suggested to be microRNA signatures of ARVC except for the miR-451a and miR-3647. Additionally, the microRNAs involved regulatory networks were accessed and the target genes of miR-21-5p and miR-135b in Wnt and Hippo pathways were preliminarily established. Several bio informatics target prediction softwares were used to predict targets of miR-21-5p and miR-135b in Wnt and Hippo signal pathway,3’UTR of YAP was shown contained potential binding sites with miR-21-5p; MoBlb and LATS2 was illuminated to be the target of miR-135b.The microRNA profiling between ARVC and healthy controls was performed, miR-21-5p and miR-135b may be involved in Wnt and Hippo signal pathway to regulate the adipogenesis. miR-21-5p was shown to be negatively correlated with YAP, and MoBlb and LATS2 was the target gene of miR-135b. Dual luciferase assay confirmed the direct target of miR-21-5p to YAP mRNA 3’UTR; and then miR-135b direct target the mRNA 3’UTR of MoBlb and LATS2. The mRNA and protein expression levels of endogenous of YAP、 MoBlb and LATS2 were both separately suppressed by miR-21-5p and miR-135b. Mover, miR-21-5p over expression significantly facilitated cardiomyocyte adipogenesis; miR-135b suppressed the myogenesis versus adipogenesis.Taken together, the present study demonstrates that miR-21-5p and miR-135b is a regulator of myogenesis versus adipogenesis in ARVC through targeting the key gene on Hippo signal pathway.ConclusionThe microRNAs profiling of ARVC heart transplantation tissues was identified a sensitive S-Poly(T) microRNA real-time polymerase chain reaction (RT-PCR) technology, several microRNAs are involved in the development of ARVC and that they might be useful as diagnostic biomarkers and therapeutic targets for ARVC. The microRNAs of miR-21-5p and miR-135b regulatory networks in Wnt and Hippo pathways were preliminarily established in our research, in vitro cell function demonstrate that miR-21-5p and miR-135b is a regulator of myogenesis versus adipo genesis in ARVC through targeting the key gene on Hippo signal pathway.