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OBJECTIVE The purpose of the study was to investigate the regulatory effect of microRNA-10-5p on TBX5 during myocardial differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats. MATERIALS AND METHODS Rat BMSCs were first isolated, cultured and identified by flow cytometry. Expression levels of BNP (Brain Natriuretic Peptide), α-actinin, and Islet-1 in BMSCs co-cultured with rat cardiomyocytes were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Dual-luciferase reporter gene assay was conducted to verify the binding condition between microRNA-10-5p and TBX5. Subsequently, we detected TBX5 expression after overexpression or knockdown of microRNA-10-5p in BMSCs. Rescue experiments were conducted by overexpression of both microRNA-10-5p and TBX5 in BMSCs, and then, the expression levels of BNP, α-actinin, and Islet-1 were detected by qRT-PCR and Western blot. RESULTS Flow cytometry results showed positive-CD73 (99.3%), positive-CD90 (95.4%), and negative-CD34 (4.2%), which were consistent with immunophenotypic characteristics of BMSCs. TBX5 overexpression or microRNA-10-5p knockdown increased mRNA levels of BNP, α-actinin, and Islet-1 in BMSCs co-cultured with rat cardiomyocytes. Dual-luciferase reporter gene assay confirmed that microRNA-10-5p could bind to TBX5. Both mRNA and protein expressions of TBX5 were negatively regulated by microRNA-10-5p. The inhibited expression levels of BNP, α-actinin, and Islet-1 by microRNA-10-5p overexpression in BMSCs were partially reversed by TBX5 overexpression. CONCLUSIONS MicroRNA-10-5p regulates BMSCs differentiation into cardiomyocytes by binding to TBX5.