Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Peking University, Beijing, China.
PLoS One. 2013 Sep 11;8(9):e74504. doi: 10.1371/journal.pone.0074504. eCollection 2013.
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.
miR-499 是一种丰富表达于心脏的 miRNA。然而，miR-499 在分化的心肌细胞或心肌细胞分化过程中的生物学功能尚不清楚。Sox6 被认为是其靶标之一，并且也被认为在心脏分化中发挥作用。因此，我们的目的是研究 Sox6 和 miR-499 在心脏分化过程中的相关性。
方法/主要发现：使用已建立的体外心肌细胞分化系统，即小鼠 P19CL6 细胞，我们发现 miR-499 在心脏分化的晚期高度表达。在稳定转染 miR-499 的细胞（P-499 细胞）中，发现 miR-499 可以促进心脏分化的早期向心肌细胞分化。值得注意的是，细胞活力测定、EdU 掺入测定和细胞周期谱分析均表明 P-499 细胞具有增生性生长的特征。进一步的研究证实，miR-499 可以促进新生大鼠心肌细胞的增殖。miR-499 敲低增强了 P19CL6 细胞在晚期分化阶段的凋亡，但 miR-499 的过表达导致凋亡率降低。Sox6 被鉴定为 miR-499 的直接靶标，并且在 P19CL6 细胞的心脏分化第 8 天或第 10 天检测到其表达。Sox6 在 P19CL6 细胞和心肌细胞中发挥作用，抑制细胞活力，促进细胞凋亡。Sox6 的过表达可以逆转 miR-499 的增殖和抗凋亡作用。还发现 miR-499 可能通过其对 Sox6 的影响来调节细胞周期蛋白 D1，从而发挥其功能。
结论/意义：miR-499 可能通过其对 Sox6 和细胞周期蛋白 D1 的影响，调节 P19CL6 细胞在心脏分化晚期的增殖和凋亡。
