miR-200c-3p regulates Epitelial-to-Mesenchymal Transition in epicardial mesothelial cells by targeting epicardial Follistatin-related protein 1

Background: 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). In particular, epicardial cells that undergo this process, acquire stem cell-like properties, migrate and differentiate into a number of distinct cell types and release paracrine factors therefore supporting cardiac repair. MicroRNAs (miRNAs) act as key regulators in EMT processes; however, the mechanisms by which miRNAs target epicardial EMT remain largely unknown. Here, we investigated the role of miRNAs as regulators of epicardial EMT and their potential targets. Methods and Results: EMT was induced in epicardial-mesothelial cells (EMCs) through TGFb1 treatment for 48, 72 and 96 hours as indicated by the expression of EMT-related genes by q-RTPCR, WB and immunofluorescence. Further, enhanced expression of stemness genes was also detected. Among several EMT-related miRNAs, miR-200c-3p expression resulted the most strongly suppressed. Interestingly, we also found a significant uregulation of Follistatin-related protein 1 (FSTL1), already identified as a potent cardiogenic factor produced by epicardial cells that promotes regeneration following MI. These results were strengthened by the detection, in vivo, of higher FSTL1 mRNA expression in epicardial cells of 3 day-infarcted hearts compared to the underlying cardiac tissue and prompted us to verify a possible involvement of FSTL1 in epicardial EMT. Bioinformatics analysis and the dual-luciferase reporter assay demonstrated that miR-200c-5p 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 TGFb1-mediated FSTL1 upregulation. Importantly, FSTL1 silencing maintained epithelial features in EMCs, despite EMT induction, and attenuated EMT-associated traits, including migration and stemness. Conclusions: Epicardial FSTL1 plays an important role in cardiac repair as a cardiogenic factor in its secreted form and by inducing EMT, stemness and migration of EMCs in a miR-200c-3p dependent pathway.