MiR-9 Promotes Angiogenesis via Targeting on Sphingosine-1- Phosphate Receptor 1

We previously demonstrated that vascular endothelial cells released VEGF-enriched exosomes to promote the tumor vasculogenesis and progression after anti-angiogenic therapies (AATs). To clarify how microRNA (miR)-9 promoted the angiogenesis of tumor-associated endothelial cells, in the present study, we investigated the association between miR-9 and sphingosine-1-phosphate (S1P) receptors in angiogenesis. The levels of miR-9 and S1P receptors in normal and tumor endothelial cells were compared with EndoDB database and their correlations were analyzed. The levels of S1P₁, S1P₂, and S1P₃ were detected in miR-9 overexpressing endothelial cells by qRT-PCR and western blot. The binding sites of miR-9 on S1P₁ and S1P₃ were predicted and tested by dual-luciferase reporter assays. Then, angiogenesis in endothelial cells overexpressing both S1P₁ and miR-9 was detected. The results showed that miR-9 is overexpressed in ECs from medulloblastoma and glioblastoma xenograft, which is negatively associated with S1P₁ and S1P₃. Overexpression of miR-9 significantly inhibited S1P₁ and S1P₃ in both mRNA and protein levels. We predicted that binding sites exist between miR-9 and S1P₁, S1P₃, but only S1P₁ was directly targeted by miR-9. Overexpression of S1P₁ significantly suppressed the miR-9-induced angiogenesis. Therefore, miR-9 induces angiogenesis via targeting on S1P₁.