MicroRNA-1-Mediated Inhibition of Cardiac Fibroblast Proliferation Through Targeting Cyclin D2 and CDK6.

Nedyalka Valkov,Hong Liu,Michelle E King,Peng Zhang,Jacob Moeller,Xiaofei Li

doi:10.3389/fcvm.2019.00065

Abstract

MicroRNA-1 (miRNA-1) has been long viewed as a muscle-specific miRNA and plays a critical role in myocardium and cardiomyocytes by controlling myocyte growth and rhythm. We identified that miRNA-1 is expressed in cardiac fibroblasts, which are one of the major non-muscle cell types in myocardium and are responsible for cardiac fibrosis in pathological conditions. In this study, we aimed to investigate the effect and mechanism of action of miRNA-1 on cardiac fibroblast proliferation. Subcutaneous angiotensin II (Ang II) infusion via osmotic minipumps for 4 weeks was used to induce myocardial interstitial fibrosis in male Sprague-Dawley rats. MiRNA-1 expression was significantly down-regulated by 68% in freshly isolated ventricular fibroblasts from Ang II-infused rats than that from control rats. Similar results were obtained in adult rat ventricular fibroblasts that were stimulated in culture by Ang II or TGFβ for 48 h. Functionally, overexpression of miRNA-1 inhibited fibroblast proliferation, whereas knockdown of endogenous miRNA-1 increased fibroblast proliferation. We then identified and validated cyclin D2 and cyclin-dependent kinase 6 (CDK6) as direct targets of miRNA-1 in cardiac fibroblasts using biochemical assays. Moreover, we showed that the inhibitory effects of miRNA-1 on cardiac fibroblast proliferation can be blunted by overexpression of its target, cyclin D2. In conclusion, our findings demonstrate miRNA-1 expression and regulation in adult ventricular fibroblasts, where it acts as a novel negative regulator of adult cardiac fibroblast proliferation that is at least partially mediated by direct targeting of two cell cycle regulators. Our results expand the understanding of the regulatory roles of miRNA-1 in cardiac cells (i.e., from myocytes to a major non-muscle cells in the heart).

Highlights

Cardiac fibroblasts are one of the most prevalent cell types in the myocardium [1] and play a key role in the maintenance of extracellular matrix in the heart [2]
We found that miRNA-1 expression was significantly downregulated by 68% in freshly isolated ventricular fibroblasts that were activated by angiotensin II (Ang II) infusion in vivo for 4 weeks (Figure 1F)
We addressed whether cyclin D2 and cyclin-dependent kinase 6 (CDK6) are direct target genes of miRNA-1 in cardiac fibroblasts using 3′-untranslated regions (UTRs) luciferase reporter assays that are widely used to determine whether a miRNA directly binds to the 3′-UTR of its putative target genes

Summary

Introduction

Cardiac fibroblasts are one of the most prevalent cell types in the myocardium [1] and play a key role in the maintenance of extracellular matrix in the heart [2]. In response to pathological stress or injury, cardiac fibroblasts are activated to proliferate quickly and produce excessive extracellular matrix proteins, which lead to cardiac fibrosis [3]. While it has long been viewed as a disease. MiRNAs are recognized as important regulators in both cardiac development and disease. A few miRNAs have been studied in cardiac fibroblasts and accumulated evidence suggest that they play an important role in regulating cardiac fibroblast signaling and function [reviewed in [18]]. MiRNA-21 was shown to regulate fibroblast survival by mediating MAPK signaling via sprouty homolog 1 [5]; miRNA-29 targets extracellular matrix proteins including collagen [19]; miRNA-30 targets connective tissue growth factor [20]

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