Figure 7. miR-16-5p directly targets VEGFA. (A) QRT-PCR analysis of VEGFA levels in postmenopausal patients with or without osteoporosis (n=10 per group). (B, C) Dual luciferase reporter assay results show firefly luciferase activity relative to Renilla luciferase activity in hMSCs transfected with dual luciferase vectors containing VEGFA-WT-3’UTR or VEGFA-MUT-3’UTR and miR-16-5p agonist (agomiR-16-5p) or negative control (agomiR-NC). (D) QRT-PCR analysis shows relative VEGFA mRNA levels in control hMSCs and hMSCs transfected with agomiR-NC, agomiR-16-5p, antagomiR-NC, and antagomiR-16-5p. (E) Western blotting analysis shows relative VEGFA protein levels expression in hMSCs transfected with siRNA-NC, and siRNA-VEGFA. (F) QRT-PCR analysis shows relative expression of osteogenic marker genes, ALP, OCN and RUNX2, in hMSCs transfected with siRNA-NC, and siRNA-VEGFA. (G, H) Alizarin red staining shows calcium deposition in hMSCs transfected with siRNA-NC, and siRNA-VEGFA for 21 days. Scale bar = 10 mm. The data are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.