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.

中文翻译：

---

Circ-100290 通过 miR-449a/eNOS 和 miR-449a/VEGFA 轴正向调节人羊膜源间充质干细胞条件培养基诱导的血管生成。

人羊膜间充质干细胞 (hAMSCs) 强大的促血管生成能力可能是一种有价值的骨再生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，我们报告了环状 RNA 100290 (circ-100290) 的表达上调和人脐静脉内皮细胞 (HUVECs) 的增强血管生成表型，与来自 hAMSCs (hAMSC-CM) 的条件培养基一起孵育，而 circ-100290 的下调逆转了亲-hAMSC-CM 诱导的 HUVEC 的血管生成能力。通过生物信息学方法预测 Circ-100290/microRNA 449a (miR-449a)/内皮一氧化氮合酶 (eNOS) 和 circ-100290/miR-449a/血管内皮生长因子 A (VEGFA) 轴，并随后通过荧光素酶报告基因检测进行验证体外。然后使用靶向 circ-100290 或过表达 circ-100290 的质粒的小干扰 RNA (siRNA) 进行功能获得或丧失检测。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，同时 eNOS 和 VEGFA 的表达降低。相比之下，circ-100290 的上调导致 hAMSC-CM 处理后 HUVEC 的管形成和迁移增强，同时 eNOS 和 VEGFA 的表达增加。此外，miR-449a 抑制剂可以在很大程度上挽救 circ-100290 沉默对 HUVEC 的影响，而 miR-449a 模拟物可以显着挽救过表达 circ-100290 对 HUVEC 的影响。使用 eNOS 或 VEGF 受体抑制剂的功能分析表明 eNOS 和 VEGFA 可能是 miR-449a 的重要靶标。最后，基质胶塞试验显示，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。我们的结果表明，circ-100290 可能通过 miR-449a/eNOS 和 miR-449a/VEGFA 轴在 hAMSC-CM 对 HUVEC 的促血管生成作用中起作用。