Figure 5

WISP-1 promotes VEGF-A expression and angiogenesis by down-regulating miR-381 expression. (a) MG-63 cells were treated with WISP-1 (30 ng/ml) for 24 h, and miR-381 expression was detected by RT-qPCR. Cells treated with 0 mg/ml of WISP-1 were used as the control. (b) MG-63 cells (normal and stable) were transfected with 3′UTR reporter assay plasmids for 24 h, then the normal cells were incubated with WISP-1 (30 ng/ml) for 24 h. The relative luciferase activity was measured. Normal cells that were not treated with WISP-1 and the stable control-shRNA cells served as the controls. (c) MG-63 cells were transfected with miR-381 mimic for 24 h, then incubated with WISP-1 (30 ng/ml) and VEGF-A expression was detected by western blot (MW, molecular weight) and ELISA. Cells transfected with control miRNA were used as the control. (d and e) MG-63 cells were transfected with miR-381 mimic for 24 h, then incubated with WISP-1 (30 ng/ml). Culture medium was collected as CM and then applied to the EPCs for 24 h. Capillary-like structure formation and migration in the EPCs was examined by tube formation (bar=100 μm) and the Transwell migration assay, respectively. CM collected from cells transfected with control miRNA was used as the control. (f) Cells were transfected with miR-381 mimic for 24 h, then incubated with WISP-1 (30 ng/ml). Culture medium was collected as CM. Chick embryos were incubated with CM for 4 days, then photographed with a stereomicroscope (bar=1 mm) and quantified by vessel count. CM collected from cells that were transfected with control miRNA were used as the control. (g and h) mRNA expression of WISP-1, VEGF-A, and miR-381 in human osteosarcoma tumor samples was examined by RT-qPCR to determine the correlation between miR-381/VEGF-A and miR-381/WISP-1. Each experiment was performed in triplicate (N=3). Results are expressed as the mean±S.E.M. *P<0.05 compared with control; ^#P<0.05 compared with the WISP-1-treated group