Candidate Non-Coding RNAs Regulate Cardiomyo-cyte Differentiation and Maturation Process

WNT and TGFβ signaling pathways play critical regulatory roles in cardiomyocyte fate determination and differentia-tion. MiRNAs are also known to regulate different biological processes and signaling pathways. Here, we intended to find candidate miRNAs that are involved in cardiac differentiation through regulation of WNT and TGFβ signaling pathways. Bio-informatics analysis suggested hsa-miR-335-3p and hsa-miR-335-5p as regulators of cardiac differentiation. Then, qRT-PCR, dual luciferase, TOP/FOP flash, and western blot analyses were done to confirm the hypothesis. Human embryonic stem cells (hESCs) were differentiated into beating cardiomyocytes, and these miRNAs showed significant expression during the differ-entiation process. Gain and loss of function of miR-335-3p and miR-335-5p resulted in BRACHYURY, GATA4, and NKX2-5 (cardiac differentiation markers) expression alteration during the course of hESC cardiac differentiation. The overexpres-sion of miR-335-3p and miR-335-5p also led to upregulation of CNX43 and TNNT2 expression, respectively. Our results sug-gest that this might be mediated through enhancement of WNT and TGFβ signaling pathways. Overall, we show that miR-335-3p/5p upregulates cardiac mesoderm (BRACHYURY) and cardiac progenitor cell (GATA4 and NKX2-5) markers, which are potentially mediated through activation of WNT and TGFβ signaling pathways. Our findings suggest miR-335-3p/5p to be considered as a regulator of the cardiac differentiation process. Keywords: miRNA, Cardiomyocyte