Figure 5.

S1P facilitates VEGF synthesis and EPC angiogenesis via the inhibition of miR-16-5p. (A) The open-source software enabled identification of miRNAs that possibly disturb the transcription of VEGF. (B) Osteoblasts were transfected with S1P. MiR-16-5p expression was determined by the qPCR assay. (C,D) The osteoblasts were transfected with miR-16-5p mimic, then stimulated by S1P. Levels of VEGF were determined by qPCR and ELISA. (E,F) Collected CM was administered to the EPCs, and angiogenesis was quantified. (G) A schematic representation of human VEGF containing the miR-16-5p binding site. (H) The luciferase plasmids with, or without miR-16-5p mimic, were transfected into osteoblasts, before stimulating them with S1P. Measurement of luciferase activity revealed VEGF promoter activity. (I) Osteoblasts were transfected with S1P₁ or S1P₃ siRNA for 24 h, or pretreated with c-Src or FAK inhibitors for30 min, then stimulated with S1P for 24 h. MiR-16-5p expression was quantified by qPCR. Results are expressed as the mean ± S.D. (n = 3). * p < 0.05 versus the control; # p < 0.05 versus S1P alone.