Figure 6. miR-150 targets EGR2 to control Ang2 generation.

A, Luciferase activity in HPAECs co-transfected with scrambled or pre-miR-150 cDNA along with WT or mutant EGR2 3′UTR luciferase reporter (n=3 experiments). B, EGR2 mRNA (left and middle panel) or protein (right panel) expression following exposure of lungs (left, n=3–4 mice) or cells to 1 mg/ml LPS (n=3 experiments). RNA was normalized against GAPDH while immunoblot with anti-actin antibody was used as a loading control. C, Ang2 expression (left) and transendothelial albumin flux (right) in HPAECs depleted of EGR2 after 1 μg/ml LPS challenge. EGR2 depletion was assessed in HPAECs after 48 h transfection with scrambled (siSc) or EGR2 siRNA (SiEGR2) (n=3 experiments). D, EMSA of control or EGR2 depleted HPAECs after LPS challenge using ³²P-labeled Ang2 oligonucleotide (n=3 experiments). E, Liposomes conjugated with either miR-150-mimic or control mimic were delivered into vasculature of mice (E, top). Forty five min later these mice were challenged with a lethal dose of LPS (40 mg/kg) and mice survival was monitored over the next 72 h. n= 8 in each group. B–C, EGR2, and Ang2 expression in mice receiving miR-150 mimic versus negative control. n=3–4. All data represent mean ± SEM. In all immunoblots, band density was quantified taking actin as the control. Survival was assessed by log rank test in panel E while in all other panels one way ANOVA and post-hoc t-test were used to compare data between groups. *P<0.05.