BACKGROUND Inflammation and oxidative stress induced by oxidized low density lipoprotein are the main causes of vascular endothelial injury and atherosclerosis. Endothelial cells are important for the formation and repair of blood vessels. However, the detailed mechanism underlying the regulation of maturity and antioxidation of stem cell-derived endothelial like cells remains unclear. Besides, YY1 and TET2 play a key role on epigenetic modifications of proliferation and differentiation of stem cells. However, the regulatory mechanism of epigenetic modification induced by YY1 and TET2 on stem cells to iECICs is also not clear. AIM Here, we want to investigate detailed mechanism underlying the regulation of maturity and antioxidation of stem cell-derived iECICs by by YY1 and TET2. METHODS The qPCR, Western blot, immunohistochemical staining and flow cytometric analysis were used to analyze the expression level of each gene. Luciferase reporter assay was used to detect the binding sites between microRNA and target genes. The hMeDIP-sequence, ChIP-PCR and dot blot were used to detect the 5-hydroxymethylcytosine modification of genomic DNA. ATP, ROS, SOD assay were used to evaluate of oxidative stress in cells. The iECICs transplantation group The ApoE-/- mice were intravenous injected of iECICs to evaluation of therapeutic effect in vivo. RESULTS Our studies have found that as the differentiation of human amniotic epithelial cells (HuAECs) is directed towards iECICs in vitro, the expression levels of vascular endothelial cell markers and miR-544 increase significantly and the expression level of YinYang 1 (YY1) decreases significantly. The luciferase reporter assay suggests that Yy1 is one of the targets of miR-544. Hydroxymethylated DNA immunoprecipitation sequencing showed that compared with HuAECs, iECICs had 174 protein-coding DNA sequences with extensive hydroxymethylation modifications. Overexpression of miR-544 inhibits the activity of the YY1/PRC2 complex and promotes the transcription and expression of the ten-eleven translocation 2 (TET2) gene, thereby activating the key factors of the serotonergic synapse pathway, CACNA1F, and CYP2D6. In addition, it promotes ability of maturity, antioxidation and vascular formation in vitro. Meanwhile, transplantation for miR-544-iECICs can significantly relieve oxidative stress injury on ApoE-/- atherosclerotic mice in vivo. CONCLUSIONS miR-544 regulates the maturity and antioxidation of iECICs derived from HuAECs by regulating the YY1/TET2/serotonergic synapse signalling axis. Video abstract.

中文翻译：

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miR-544 通过调节 YY1/TET2 信号轴促进干细胞衍生的内皮样细胞的成熟和抗氧化。

背景氧化低密度脂蛋白引起的炎症和氧化应激是血管内皮损伤和动脉粥样硬化的主要原因。内皮细胞对于血管的形成和修复很重要。然而，干细胞衍生的内皮样细胞成熟和抗氧化调节的详细机制仍不清楚。此外，YY1和TET2在干细胞增殖和分化的表观遗传修饰中起关键作用。然而，YY1和TET2对干细胞的表观遗传修饰对iECICs的调控机制也不清楚。目的 在这里，我们想研究 YY1 和 TET2 调控干细胞衍生 iECICs 成熟和抗氧化的详细机制。方法 qPCR、蛋白质印迹、采用免疫组化染色和流式细胞术分析各基因的表达水平。荧光素酶报告基因检测用于检测微小RNA与靶基因之间的结合位点。hMeDIP 序列、ChIP-PCR 和斑点印迹用于检测基因组 DNA 的 5-羟甲基胞嘧啶修饰。ATP、ROS、SOD测定用于评估细胞中的氧化应激。iECICs移植组对ApoE-/-小鼠静脉注射iECICs以评价体内治疗效果。结果 我们的研究发现，随着体外人羊膜上皮细胞（HuAECs）向iECICs的分化，血管内皮细胞标志物和miR-544的表达水平显着增加，阴阳1（YY1）的表达水平显着降低. 荧光素酶报告基因检测表明 Yy1 是 miR-544 的靶标之一。羟甲基化 DNA 免疫沉淀测序表明，与 HuAECs 相比，iECICs 有 174 个蛋白质编码 DNA 序列，具有广泛的羟甲基化修饰。miR-544的过表达抑制了YY1/PRC2复合物的活性，促进了10-11易位2（TET2）基因的转录和表达，从而激活了5-羟色胺能突触通路的关键因子CACNA1F和CYP2D6。此外，它还促进体外成熟、抗氧化和血管形成的能力。同时，移植miR-544-iECICs可以显着减轻体内ApoE-/-动脉粥样硬化小鼠的氧化应激损伤。结论 miR-544 通过调节 YY1/TET2/5-羟色胺能突触信号轴来调节源自 HuAECs 的 iECICs 的成熟和抗氧化。视频摘要。