miR-194-3p regulates epithelial-mesenchymal transition in embryonic epicardial cells via p120/β-catenin signaling

Abstract

The epicardium is integral to cardiac development and facilitates endogenous heart regeneration and repair. While miR-194-3p is associated with cellular migration and invasion, its impact on epicardial cells remains uncharted. In this work we use gain-of-function and loss-of-function methodologies to investigate the function of miR-194-3p in cardiac development. We culture embryonic epicardial cells in vitro and subject them to transforming growth factor β (TGF-β) treatment to induce epithelial-mesenchymal transition (EMT) and monitor miR-194-3p expression. In addition, the effects of miR-194-3p mimics and inhibitors on epicardial cell development and changes in EMT are investigated. To validate the binding targets of miR-194-3p and its ability to recover the target gene-phenotype, we produce a mutant vector p120-catenin-3′UTR-MUT. In epicardial cells, TGF-β-induced EMT results in a notable overexpression of miR-194-3p. The administration of miR-194-3p mimics promotes EMT, which is correlated with elevated levels of mesenchymal markers. Conversely, miR-194-3p inhibitor attenuates EMT. Further investigations reveal a negative correlation between miR-194-3p and p120-catenin, which influences β-catenin level in the cell adhesion pathway. The suppression of EMT caused by the miR-194-3p inhibitor is balanced by silencing of p120-catenin. In conclusion, miR-194-3p directly targets p120-catenin and modulates its expression, which in turn alters β-catenin expression, critically influencing the EMT process in the embryonic epicardial cells via the cell adhesion mechanism.

1. Introduction

Cardiovascular illnesses continue to be one of the most serious global health threats, endangering the lives of millions of people. Central to the heart’s function is the epicardium, an integral component that plays an essential role in cardiac development and endogenous regenerative processes [1]. A single layer of precursor epicardial cells develops into smooth muscle and fibroblasts, which in turn form cardiac endothelial cells and interstitial cells [2,3]. By embryonic day (E) 9.5 in mice, precursor epicardial cells attach to the myocardium and crease in the epithelial layer to form a single cell layer that envelops the entire myocardium [4]. By E11.5, the heart is fully covered by epicardial cells, exhibiting a cuboidal epithelial phenotype [5]. Membrane adhesion molecules such as β-catenin and E-cadherin are expressed to preserve the integrity and top-to-base polarity of the epicardial cell layer [6]. Numerous genes encoding transcription factors, such as Wilms tumor 1 (Wt1) [7], transcription factor 21 (Tcf21) [8], and T-box transcription factor 18 (Tbx18) [9], are expressed in the epicardium.

Epithelial-mesenchymal transition (EMT), which occurs as the epicardium differentiates, is followed by stratification, which results in the formation of cells derived from the epicardium that span the gap between the epicardium and myocardium. These cells form the sub-epicardial mesenchyme and infiltrate the myocardium, where they evolve into supporting cells of the heart [10‒13]. TGF-β is among the most extensively studied upstream regulatory molecules for EMT and extracellular matrix (ECM) production [14]. TGF-β1–3 isoforms are expressed in the epicardium of mice at E12.5, and they bind to the same receptor complex, collectively inducing epithelial cell EMT [15‒18]. The transition of primary epithelial tissue into highly motile mesenchymal cells is a characteristic feature of the development of multicellular organisms. It promotes the growth and development of organisms by enabling epithelial cells to migrate inside the ECM and build tissues at particular sites [11]. Notably, the molecular mechanisms underlying the differentiation of epicardium-derived cells into distinct cell types during cardiac development remain elusive. Interestingly, adult epicardial cells are mostly quiescent, but when they are subjected to damage, they become active and aid in repair by re-expressing the adult heart’s developmental programs [19].

MicroRNAs, comprising 19–25 nucleotides, constitute a vital subset of small noncoding RNAs pivotal in post-transcriptional regulation of gene expression by targeting the 3′UTR of coding RNAs, resulting in the inhibition of protein translation and facilitation of mRNA degradation [20]. The essential involvement of miR-194-3p in cellular migratory and invasive activities has been shown in recent scientific reports [21‒23]. However, the precise modulatory role of miR-194-3p in the EMT of the epicardium during cardiac embryogenesis still needs to be addressed. Notably, while many in vitro models have been established to investigate epicardial EMT [24,25], the regulatory orchestration of microRNAs, especially that of miR-194-3p, in this physiological transition has yet to be explored. Based on our research, we propose that miR-194-3p plays a crucial role in mediating epicardial EMT, possibly by influencing the cell adhesion signaling pathway.