Circ-100290 Positively Regulates Angiogenesis Induced by Conditioned Medium of Human Amnion-Derived Mesenchymal Stem Cells Through miR-449a/eNOS and miR-449a/VEGFA Axes
Circ-100290 Positively Regulates Angiogenesis Induced by Conditioned Medium of Human Amnion-Derived Mesenchymal Stem Cells Through miR-449a/eNOS and miR-449a/VEGFA Axes
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Circ-100290 通过 miR-449a/eNOS 和 miR-449a/VEGFA 轴正向调节人羊膜来源间充质干细胞条件培养基诱导的血管生成
DOI：
10.7150/ijbs.39895
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发表时间：
2020-01-01
影响因子：
9.2
通讯作者：
Yuan, Xiaoqin
中科院分区：
文献类型：
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作者：
Tang, Zichun;Wu, Xiaoyue;Yuan, Xiaoqin
人骨髓间充质干细胞（hAMSCs）强大的促血管生成能力可能是骨再生的有价值的治疗性血管生成策略。然而，这一过程背后的分子机制在很大程度上仍然未知。在此，我们报告了用来自hAMSC的条件培养基（hAMSC-CM）孵育的人脐静脉内皮细胞（HUVECs）的环状RNA 100290（circ-100290）的上调表达和增强的血管生成表型，而circ-100290的下调逆转了hAMSC-CM诱导的HUVECs的促血管生成能力。通过生物信息学方法预测Circ-100290/microRNA 449 a（miR-449 a）/内皮型一氧化氮合酶（eNOS）和circ-100290/miR-449 a/血管内皮生长因子A（VEGFA）轴，随后通过体外荧光素酶报告基因测定进行验证。然后使用靶向circ-100290的小干扰RNA（siRNA）或过表达circ-100290的质粒进行功能获得或丧失试验。正如预期的那样，HUVEC中circ-100290的下调导致hAMSC-CM处理后HUVEC的管形成和迁移减弱，沿着eNOS和VEGFA的表达降低。相反，circ-100290的上调导致hAMSC-CM处理后HUVEC的管形成和迁移增强，沿着eNOS和VEGFA表达增加。此外，miR-449 a抑制剂可以在很大程度上挽救circ-100290沉默对HUVEC的作用，而miR-449 a模拟物可以显著挽救过表达circ-100290对HUVEC的作用。使用eNOS或VEGF受体抑制剂的功能测定表明eNOS和VEGFA可能是miR-449 a的重要靶点。最后，基质胶塞测定显示，当circ-100290在HUVEC中沉默时，血管生成减弱，但当circ-100290在体内过表达时，血管生成增强。我们的研究结果表明，circ-100290可能通过miR-449 a/eNOS和miR-449 a/VEGFA轴在hAMSC-CM对HUVEC的促血管生成作用中发挥作用。
The powerful pro-angiogenic capacity of human amnion-derived mesenchymal stem cells (hAMSCs) could be a valuable therapeutic angiogenesis strategy for bone regeneration. However, the molecular mechanisms underlying this process remain largely unknown. Herein, we report upregulated expression of circular RNA 100290 (circ-100290) and an enhanced angiogenic phenotype of human umbilical vein endothelial cells (HUVECs) incubated with conditioned medium from hAMSCs (hAMSC-CM), whereas downregulation of circ-100290 reversed the pro-angiogenic capacity of HUVECs induced by hAMSC-CM. Circ-100290/microRNA 449a (miR-449a)/endothelial nitric oxide synthase (eNOS) and circ-100290/miR-449a/vascular endothelial growth factor A (VEGFA) axes were predicted by a bioinformatics method and subsequently verified by luciferase reporter assays in vitro. Gain- or loss-of-function assays were then performed using small interfering RNAs (siRNAs) targeting circ-100290, or a plasmid overexpressing circ-100290. As expected, downregulation of circ-100290 in HUVECs led to weakened tube formation and migration of HUVECs following hAMSC-CM treatment, along with decreased expression of eNOS and VEGFA. In contrast, upregulation of circ-100290 led to enhanced tube formation and migration of HUVECs following hAMSC-CM treatment, along with increased expression of eNOS and VEGFA. Furthermore, a miR-449a inhibitor could largely rescue the effect of circ-100290 silencing on HUVECs, whereas a miR-449a mimic could significantly rescue the effect of overexpressing circ-100290 on HUVECs. Functional assays using eNOS or VEGF receptor inhibitors indicated eNOS and VEGFA may be important targets of miR-449a. Finally, a Matrigel plug assay revealed weakened angiogenesis when circ-100290 was silenced in HUVECs, but enhanced angiogenesis when circ-100290 was overexpressed in vivo. Our results suggest that circ-100290 might function via miR-449a/eNOS and miR-449a/VEGFA axes in the pro-angiogenic role of hAMSC-CM on HUVECs.