Zhang Rui, Wang Nan, Zhang Li-Nan, Huang Na, Song Tie-Feng, Li Zheng-Zheng, Li Man, Luo Xue-Gang, Zhou Hao, He Hong-Peng, Zhang Xiao-Yu, Ma Wenjian, Zhang Tong-Cun
Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, People's Republic of China.
Institute of Biology and Medicine, Wuhan University of Science and Technology, Wuhan, People's Republic of China.
Stem Cells. 2016 May;34(5):1273-83. doi: 10.1002/stem.2288. Epub 2016 Feb 13.
Human mesenchymal stem cells (hMSCs) possess the potential to differentiate into endothelial cells (EC). DNA methylation plays an important role in cell differentiation during development. However, the role of the DNA methyltransferases Dnmt1 and Dnmt3a in specific arterial differentiation of hMSCs is not clear. Here, we show that the CpG islands in the promoter regions of the EC specification and arterial marker genes were highly methylated in hMSCs based on bisulfite genomic sequencing. Treatment with the DNMT inhibitor 5-aza-dc induced the reactivation of EC specification and arterial marker genes by promoting demethylation of these genes as well as stimulating tube-like structure formation. The hMSCs with stable knockdown of Dnmt1/Dnmt3a were highly angiogenic and expressed several arterial specific transcription factors and marker genes. A Matrigel plug assay confirmed that Dnmt1/Dnmt3a stable knockdown hMSCs enhanced blood vessel formation compared with WT MSCs. We also identified that the transcription factor E2F1 could upregulate the transcription of arterial marker genes by binding to the promoters of arterial genes, suggesting its critical role for arterial specification. Moreover, miRNA gain/loss-of-function analyses revealed that miR152 and miR30a were involved in endothelial differentiation of hMSCs by targeting Dnmt1 and Dnmt3a, respectively. Taken together, these data suggest that Dnmt1 and Dnmt3a are critical regulators for epigenetic silencing of EC marker genes and that E2F1 plays an important role in promoting arterial cell determination. Stem Cells 2016;34:1273-1283.
人间充质干细胞（hMSCs）具有分化为内皮细胞（EC）的潜力。DNA甲基化在发育过程中的细胞分化中起着重要作用。然而，DNA甲基转移酶Dnmt1和Dnmt3a在hMSCs特定动脉分化中的作用尚不清楚。在此，我们通过亚硫酸氢盐基因组测序表明，hMSCs中EC特异性和动脉标记基因启动子区域的CpG岛高度甲基化。用DNMT抑制剂5-氮杂-2'-脱氧胞苷处理可通过促进这些基因的去甲基化以及刺激管状结构形成来诱导EC特异性和动脉标记基因的重新激活。稳定敲低Dnmt1/Dnmt3a的hMSCs具有高度血管生成性，并表达几种动脉特异性转录因子和标记基因。基质胶栓试验证实，与野生型MSCs相比，稳定敲低Dnmt1/Dnmt3a的hMSCs增强了血管形成。我们还发现转录因子E2F1可通过结合动脉基因的启动子来上调动脉标记基因的转录，表明其在动脉特化中起关键作用。此外，miRNA功能获得/丧失分析表明，miR152和miR30a分别通过靶向Dnmt1和Dnmt3a参与hMSCs的内皮分化。综上所述，这些数据表明Dnmt1和Dnmt3a是EC标记基因表观遗传沉默的关键调节因子，并且E2F1在促进动脉细胞决定中起重要作用。《干细胞》2016年；34卷：1273 - 1283页
