Abstract
BackgroundMiR-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 FindingsUsing 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/SignificancemiR-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.
Highlights
Heart morphogenesis and development is a complicated process, in which cell cycle progression/exit control is of paramount importance
By gain- and loss-of-function methods, including stable overexpression of miR-499 and Sox6 and transient downregulation of miR-499 and Sox6, we demonstrate that Sox6, as a repressor of cyclin D1, arrests cardiomyocyte proliferation and facilitates cell cycle exit; miR-499 on the other hand downregulates the expression of its target protein, Sox6, to an appropriate level so as to prevent cardiomyocyte apoptosis
When P19CL6 cells were treated with 1% dimethyl sulfoxide (DMSO) for 12 days, over 95% of the cells expressed sarcomeric α-actinin, a marker for mature cardiomyocytes, and rhythmic spontaneous beating started (Figure 1A, Video S1)
Summary
Heart morphogenesis and development is a complicated process, in which cell cycle progression/exit control is of paramount importance. During the embryonic and fetal stages, cardiomyocytes rapidly proliferate so that a sufficient number of cells are produced to form the myocardium [1]. PLOS ONE | www.plosone.org miR-499 Targets Sox in Cardiac Differentiation cardiomyocytes complete terminal differentiation and the cell cycle is permanently arrested [5,6]. This phenomenon is common, but details of the mechanisms are currently not very clear. The biological functions of miR-499 in differentiated cardiomyocytes or in the cardiomyocyte differentiation process is not very clear. Our aim was to investigate the association between Sox and miR-499 during cardiac differentiation
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