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Fig. 4. Regulation of p85β expression by miR-126. (A) Quantitative PCR analysis confirms the almost complete absence of miR-126 expression in HUVEC transfected with a miR-126 hairpin inhibitor, as opposed to a scrambled control (scr). (B) Impaired migration of HUVEC transfected with the miR-126 hairpin inhibitor, versus scr, in the in vitro scratch wound assay (^*P<0.05 versus scrambled inhibitor-transfected). (C) Impaired VEGF-dependent Akt and Erk phosphorylation in HUVEC transfected with the miR-126 hairpin inhibitor, versus scr. (D) Target site alignment for miR-126 in the 3'UTR of Pik3r2, which encodes p85β. (E) p85β (Pik3r2) is a direct target of miR-126 as shown by dose-dependent repression by miR-126 of luciferase expression from the wild-type p85β 3'UTR, but not the control Lin41 3'UTR, reporters in 293T cells. Mutation of the miR-126 binding site in the p85β 3'UTR (p85β mut) abrogates repression by miR-126, identifying p85β as a direct target. ^*P<0.05 versus no miR-126 expression vector and P<0.05 versus p85β mut and Lin41 3'UTR reporter construct, for a given dose of miR-126 expression vector (0, 10 and 100 ng). NS, not significant. (F) p85 is upregulated in primary brain endothelial cells isolated from miR-126^/, but not Egfl7^/, mice as assessed by western blot with anti-pan p85 antibody; anti-actin antibody provided a loading control. (G) Upregulation of p85β expression in HUVEC transfected with a hairpin inhibitor targeting miR-126, versus scr. (H) Adenoviral expression of p85β in HUVEC is sufficient to inhibit VEGF-induced Akt phosphorylation.

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