Fig 2: Associations between S1P1, S1P3, and miR-9 were detected by dual-luciferase reporter assay. The structures of S1P1 mRNA (transcript variant 1 M_001400.5, and transcript variant 2 NM_001320730.1; the variant 2 differs in the 5'UTR compared to variant 1, both variants 1 and 2 encode the same protein) and S1P3 mRNA (NM_005226.4) including coding DNA sequence (CDS) and untranslated regions were depicted. The blue band indicated the putative miR-9 binding sequence in the target mRNA molecules. The binding sites located at 3'UTR were screened for Dual-Luciferase Reporter Assay analysis. (A) Putative wild-type (WT) binding sequence and its mutation (MUT) sequence in the 3'UTR of S1P1. Luciferase activity was measured. (B) Putative WT binding sequence and its MUT sequence in the 3'UTR of S1P3. Luciferase activity was measured. NC: miR-9 mimics negative control. Mean ± SEM. n = 3, ***P < 0.001.

Fig 3: Relationship between miR-9 and S1P receptors. The correlation between the significant differentially expressed miR-9 loci and specific S1P receptors in normal ECs (NEC) and tumor ECs (TEC) were performed. (A) Correlation between miR-9-2 and S1P receptors in Wnt-medulloblastoma; (B) correlation between miR-9-1, miR-9-2, and S1P2 in Shh-medulloblastoma. Then, the levels of S1P receptors in HUVECs upon miR-9 mimics transfection were detected by qRT-PCR. (C) miR-9 levels in HUVECs overexpressing miR-9 (left) and mRNA expression of S1P1, S1P2, and S1P3 (right). NC: miR-9 mimics negative control. Mean ± SEM. n = 4, *P < 0.05, **P < 0.01, ***P < 0.001.