MicroRNA-1 and microRNA-499 directly reprogram cardiac myofibroblasts into cardiomyocyte-like cells

Zhang Cheng; Zhang Shao-zhong; Zhang Ya-zhou; Zhang Guo-heng; Wang Yun-ru; Dong Hong-yan; Zhang Zhong-ming

doi:10.3969/j.issn.2095-4344.2013.45.016

Abstract：BACKGROUND:MicroRNAs are a class of smal non-coding RNA molecules. MicroRNA is involved in the regulation of gene expression, and plays an important role in promoting the proliferation and differentiation of precursor cells. Whether microRNAs can promote progenitor cells to differentiate into myocardial cells has been rarely reported. <br> OBJECTIVE:To investigate the feasibility of direct reprogramming of neonatal mouse cardiac myofibroblasts to cardiomyocyte-like cells by specific microRNAs (microRNA-1 and microRNA-499). <br> METHODS:Cardiac fibroblasts isolated from neonatal mice were cultured in vitro at 37 ℃ under normal oxygen, cells were used at the second passage for the fol owing experiments, and then cultured at 37 ℃ in 3%O2 for 48 hours. Immunofluorescent staining was used to detect expression of vimentin which is a specific marker for Cardiac fibroblasts. Immunofluorescent staining and flow cytometry assay were employed to detect expression ofα-smooth muscle actin which is a specific marker for cardiac myofibroblasts. There were four groups in the experiment:microRNA-1 group, microRNA-499 group, microRNA-1/microRNA-499 co-tranfection group, and blank control group. MicroRNA profile between cardiac myofibroblasts and myocardial cells was determined by microarray analysis via the miRCURYTM Array microarray kit. MicroRNA microarray results were validated by real-time quantitative PCR. <br> RESULTS AND CONCLUSION:After overexpression of microRNA-1 and microRNA-499 in cardiac myofibroblasts, specific factors of heart development at different stages had different expressions;compared with the blank control group, the expression of GATA4, Tbx5, Mef-2c,α-MHC increased significantly in the remaining three groups, but Mesp1, Isl1 expression was not significantly changed). The results provide strong evidence for the capacity of microRNAs to induce expression of cardiomyocyte markers in cardiac myofibroblasts and demonstrate the potential of specific microRNAs that can direct reprogram cardiac myofibroblasts to cardiomyocytes in vitro.