MiR-499 Regulates Cell Proliferation and Apoptosis during Late-Stage Cardiac Differentiation via Sox6 and Cyclin D1

BACKGROUND: MiR-499 is a cardiac-abundant miRNA. However, the biological functions of miR-499 in differentiated cardiomyocytes or in the cardiomyocyte differentiation process is not very clear. Sox6 is believed to be one of its targets, and is also believed to play a role in cardiac differentiation. Therefore, our aim was to investigate the association between Sox6 and miR-499 during cardiac differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Using a well-established in vitro cardiomyocyte differentiation system, mouse P19CL6 cells, we found that miR-499 was highly expressed in the late stage of cardiac differentiation. In cells stably transfected with miR-499 (P-499 cells), it was found that miR-499 could promote the differentiation into cardiomyocytes at the early stage of cardiac differentiation. Notably, cell viability assay, EdU incorporation assay, and cell cycle profile analysis all showed that the P-499 cells displayed the distinctive feature of hyperplastic growth. Further investigation confirmed that miR-499 could promote neonatal rat cardiomyocyte proliferation. MiR-499 knock-down enhanced apoptosis in the late differentiation stage in P19CL6 cells, but overexpression of miR-499 resulted in a decrease in the apoptosis rate. Sox6 was identified as a direct target of miR-499 and its expression was detected from day 8 or day 10 of cardiac differentiation of P19CL6 cells. Sox6 played a role in cell viability, inhibited cell proliferation and promoted cell apoptosis in P19CL6 cells and cardiomyocytes. The overexpression of Sox6 could reverse the proliferation and anti-apoptosis effects of miR-499. It was also found that miR-499 might exert its function by regulating cyclin D1 via its influence on Sox6. CONCLUSIONS/SIGNIFICANCE: miR-499 probably regulates the proliferation and apoptosis of P19CL6 cells in the late stage of cardiac differentiation via its effects on Sox6 and cyclin D1.