MiR-200c-3p Regulates Epitelial-to-Mesenchymal Transition in Epicardial Mesothelial Cells by Targeting Epicardial Follistatin-Related Protein 1.

Elena Pontemezzo,Federica Limana,Alessandra Magenta,Elena Astanina,Sara Sileno,Eleonora Foglio,Marco D’Agostino,Antonia Germani,Laura Pellegrini,Matteo Antonio Russo,Enza Vernucci,Federico Bussolino

doi:10.3390/ijms22094971

Abstract

Recent findings suggest that epithelial to mesenchymal transition (EMT), a key step during heart development, is involved in cardiac tissue repair following myocardial infarction (MI). MicroRNAs (miRNAs) act as key regulators in EMT processes; however, the mechanisms by which miRNAs target epicardial EMT remain largely unknown. Here, by using an in vitro model of epicardial EMT, we investigated the role of miRNAs as regulators of this process and their potential targets. EMT was induced in murine epicardial-mesothelial cells (EMCs) through TGF β1 treatment for 48, 72, and 96 h as indicated by the expression of EMT-related genes by qRT-PCR, WB, and immunofluorescence. Further, enhanced expression of stemness genes was also detected. Among several EMT-related miRNAs, miR-200c-3p expression resulted as the most strongly suppressed. Interestingly, we also found a significant upregulation of Follistatin-related protein 1 (FSTL1), a miR-200c predicted target already identified as a potent cardiogenic factor produced by epicardial cells that promotes regeneration following MI. Dual-luciferase reporter assay demonstrated that miR-200c-3p directly targeted the 3′-untranslated region of FSTL1 in EMCs. Consistently, WB analysis showed that knockdown of miR-200c-3p significantly increased FSTL1 expression, whereas overexpression of miR-200c-3p counteracted TGF β1-mediated FSTL1 upregulation. Importantly, FSTL1 silencing maintained epithelial features in EMCs, despite EMT induction by TGF β1, and attenuated EMT-associated traits, including migration and stemness. In conclusion, epicardial FSTL1, an important cardiogenic factor in its secreted form, induces EMT, stemness, and migration of EMCs in a miR-200c-3p dependent pathway.

Highlights

Emerging evidence suggests that epithelial to mesenchymal transition (EMT) might contribute directly to stem cell phenotypes both in cancer stem cells and in stem cells from adult tissues, including the heart [1,2,3]
As an initial step toward identifying miRNAs that may be implicated in TGF β1induced EMT of epicardial mesothelial cells (EMCs), we focused on the expression of several miRNAs mainly involved in epicardial EMT [11,12,13], and in the EMT of other cell types [10] (Figure 5A,B)
We have identified Follistatin-related protein 1 (FSTL1) as a potential EMT regulator of EMC

Summary

Introduction

Emerging evidence suggests that epithelial to mesenchymal transition (EMT) might contribute directly to stem cell phenotypes both in cancer stem cells and in stem cells from adult tissues, including the heart [1,2,3]. In the adult murine heart, following infarction, epicardial cells undergo EMT, giving rise to c-kit+ cells whose differentiation into the main cardiac cell lineages is induced by activation of fetal epicardial genes [6]. These results were supported by another more recent study demonstrating that exosomal clusterin, isolated from the pericardial fluid of patients with acute MI, improved cardiac function in mice following MI in part by promoting the formation of c-kit+ cells through epicardial EMT [3].

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