Figure 6

MiR-132 regulates Cdh5 expression and brain vascular integrity by directly targeting endothelial eef2k. (A) Schematic showing two potential miR-132 binding sites (in green) in the eef2k3′ UTR. Two mutants (“Mut-1”, “Mut-2”) of the eef2k3′ UTR were introduced by replacing the wildtype binding sequence (GACTGTT) with a mutant sequence (AGTCTCC, in red) at the two corresponding binding sites. The seed sequence of miR-132 is in blue. (B) Suppression of the luciferase activity of the eef2k3′ UTR reporter in HEK293 cells by miR-132. MiR-132 sensor served as a positive control. The experiments were repeated three times. (C) Requirement of the binding site 2 in the eef2k3′ UTR for miR-132 effect. The experiments were repeated three times. (D, E) Effects of miR-132 overexpression (D) or knockdown (E) on eEF2K expression in zebrafish larvae. The experiments were repeated three times. (F) Pharmacological evidence showing eEF2K mediates the intracranial hemorrhage effect of miR-132 knockdown. NH125, eEF2K inhibitor; rapamycin, eEF2K non-specific activator. The experiments were repeated 5-8 times, and for each time, > 15 embryos were examined for each group. (G-I) Rescue effect of eef2k knockdown on miR-132 knockdown-induced intracranial hemorrhage (G), DAPI leakage in the brain (H) and Cdh5 expression reduction (I) in zebrafish larvae. For G, the experiments were repeated 6-7 times, and for each time, > 53 embryos were examined for each group. For H, 8, 8, and 11 embryos were analyzed for “Ctrl MO”, “miR-132 MO” and “miR-132 MO + eef2k MO”, respectively. For I, the experiments were repeated four times. (J) Effect of neuron-specific miR-132 downregulation on endothelial eEF2K expression in zebrafish larvae. ECs were sorted by flow cytometry from 3-dpf Tg(Flk1:eGFP) larvae, which were injected with HuC:tdT or HuC:tdT-miR-132-S plasmid. The experiments were repeated three times. (K-M) Effects of EC-specific Eef2k overexpression on intracranial hemorrhage (K), DAPI leakage in the brain (L), and Cdh5 expression (M) of zebrafish larvae. The Eef2k was cloned into Flk1:tdT-P2A vector (“Flk1:tdT-P2A-Eef2k”). Expression of tdT in ECs (“Flk1:tdT”) served as a control. For K, the experiments were repeated nine times, and for each time, > 5 embryos were examined for each group. For L, 12 and 13 embryos were analyzed for “Flk1:tdT” and “Flk1:tdT-P2A-Eef2k”, respectively. For M, the experiments were repeated six times. Error bars, SEM. n.s., no significant; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (unpaired two-tailed Student's t-test for B, D, E, J-M; one-way ANOVA with post hoc Tukey's multiple comparison test for C and I; one-way ANOVA with post hoc Bonferroni's multiple comparison test for F, G and H).