Figure 2.

miR-93-5p Negatively Affects VEGFA Expression

(A) Putative binding sites of miR-17 family members including miR-17-5p, miR-20a-5p, and miR-93-5p in the 3′-UTR of VEGFA mRNA. (B) Generation of the VEGFA 3′-UTR wild-type and mutant constructs by cloning into the psiCHECK-2 plasmid. (C) Dual-luciferase reporter assays showing that miR-93-5p significantly reduced luciferase activity of VEGFA 3′-UTR but not the luciferase activity of the mutant VEGFA 3′-UTR. The Renilla luciferase activity was normalized to the firefly luciferase activity, and data were presented as mean ± SD from three independent experiments. (D) qPCR determining the level of miR-93-5p expression in hBMECs transfected with an miR-93-5p mimic or inhibitor. U6 was used as internal reference, and data were presented as mean ± SD from three independent experiments. (E) qPCR determining the effects of transfection with an miR-93-5p mimic or inhibitor on the expression of VEGFA. GAPDH was used as internal reference, and data were presented as mean ± SD from three independent experiments. (F) Western blot analysis of VEGFA expression in hBMECs treated with miR-93-5p mimic or inhibitor. β-actin was used as loading control, and densitometry was performed to quantify differences. (G) Levels of secretory VEGFA in culture supernatant of hBMECs transfected with miR-93-5p mimic or inhibitor, as determined by ELISA. Results were calculated as mean ± SD from three independent experiments.