Key Laboratory of Basic Research in Cardiology of Ministry of Education of China, East Hospital, Tongji University School of Medicine, 200120 Shanghai, China.
J Mol Cell Cardiol. 2012 Dec;53(6):751-9. doi: 10.1016/j.yjmcc.2012.08.024. Epub 2012 Sep 7.
Human cardiomyocyte progenitor cells (hCMPCs) are cardiac progenitor cells that are unique for their efficient differentiation into beating cardiomyocytes without requiring co-culture with neonatal cardiomyocytes. hCMPCs have shown great potential in preserving the function of infarcted mouse myocardium. MiRNA-204 has been reported to be up-regulated in differentiated hCMPCs, however, its biological significance is unclear. In this study, hCMPC proliferation, viability, apoptosis and necrosis were determined using the ELISA Kit (colorimetric BrdU detection), Cell Counting Kit-8, and Annexin V and propidium iodide staining, respectively. MiRNA-204 inhibition promoted hCMPC proliferation without affecting cell viability and the level of apoptosis and necrosis, indicating that miRNA-204 might be required for hCMPC differentiation. Quantitative reverse transcriptase-polymerase chain reactions were used to detect the expression profile of cardiac genes, including MEF2C, GATA-4, Nkx-2.5, TropT, βMHC, and cActin. Cardiac α-actin staining was used to quantify the degree of differentiation. MiRNA-204 inhibition significantly down-regulated TropT, βMHC, and cActin and reduced differentiation by 47.81% after 2 weeks of differentiation induction. Interestingly, miRNA-204 mimics (30 nM) did not promote hCMPC proliferation and differentiation. The bioinformatic tool GOmir identified the activating transcription factor 2 (ATF-2) as a potential target, which was confirmed by Western blot and a luciferase reporter assay. ATF-2 overexpression promoted hCMPC proliferation, further demonstrating the role played by ATF-2 as a target gene of miRNA-204. Therefore, miRNA-204 is required for hCMPC differentiation and ATF-2 is a target gene of miRNA-204 in hCMPCs. This study indicates that miRNA-204 is among the regulators that drive hCMPC proliferation and differentiation, and miRNA-204 might be used to influence cell fate.
人心肌细胞祖细胞（hCMPCs）是一种独特的心肌前体细胞，能够高效分化为搏动的心肌细胞，而无需与新生心肌细胞共培养。hCMPCs 在保护梗死小鼠心肌功能方面显示出巨大的潜力。据报道，miRNA-204 在分化的 hCMPCs 中上调，但它的生物学意义尚不清楚。在这项研究中，使用 ELISA 试剂盒（比色 BrdU 检测）、细胞计数试剂盒-8、Annexin V 和碘化丙啶染色分别测定 hCMPC 的增殖、活力、凋亡和坏死。miRNA-204 抑制促进 hCMPC 增殖，而不影响细胞活力以及凋亡和坏死水平，表明 miRNA-204 可能是 hCMPC 分化所必需的。定量逆转录-聚合酶链反应用于检测包括 MEF2C、GATA-4、Nkx-2.5、TropT、βMHC 和 cActin 在内的心脏基因的表达谱。心肌α-肌动蛋白染色用于量化分化程度。miRNA-204 抑制显著下调 TropT、βMHC 和 cActin，并在分化诱导 2 周后使分化减少 47.81%。有趣的是，miRNA-204 模拟物（30 nM）并没有促进 hCMPC 的增殖和分化。生物信息学工具 GOmir 鉴定出激活转录因子 2（ATF-2）作为一个潜在的靶点，该靶点通过 Western blot 和荧光素酶报告基因检测得到了验证。ATF-2 过表达促进 hCMPC 增殖，进一步证明了 ATF-2 作为 miRNA-204 的靶基因的作用。因此，miRNA-204 是 hCMPC 分化所必需的，而 ATF-2 是 hCMPC 中 miRNA-204 的靶基因。这项研究表明，miRNA-204 是驱动 hCMPC 增殖和分化的调控因子之一，miRNA-204 可能被用于影响细胞命运。
