Int J Biol Sci 2020; 16(12):2131-2144. doi:10.7150/ijbs.39895

Research Paper

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

Zichun Tang1,2, Xiaoyue Wu2, Liping Hu2, Yijing Xiao2, Junling Tan2, Siyu Zuo4, Ming Shen2,3✉, Xiaoqin Yuan4✉

1. Department of Stomatology, Tongling People's Hospital, Tongling, China
2. Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, China
3. Department of Dental Implant, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing, China
4. Nanjing Medical University, Department of Anatomy, Histology and Embryology, Nanjing, China
Zichun Tang and Xiaoyue Wu contributed equally to this work.

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Citation:
Tang Z, Wu X, Hu L, Xiao Y, Tan J, Zuo S, Shen M, Yuan X. 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. Int J Biol Sci 2020; 16(12):2131-2144. doi:10.7150/ijbs.39895. Available from http://www.ijbs.com/v16p2131.htm

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Abstract

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.

Keywords: circular RNA 100290, amnion-derived mesenchymal stem cells, angiogenesis, human umbilical vein endothelial cells, eNOS, VEGFA