FIG. 7.

miR-711 downregulates expression of Ang-1. (A) qPCR for miR-711 in cortex and hippocampus after TBI and etoposide (Etop) treatment of RCN was performed as described in Figure 1A,B. (B) miR-711 mimic downregulates Ang-1 expression. RCNs were transfected with miR-711, negative control (-ve Con) mimics, or mock transfected. Neurons were harvested 24 h after transfection. Whole-cell lysates were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against Ang-1 and β-actin. Protein levels were quantified by densitometry, normalized to β-actin, and presented as fold change compared to control untreated levels. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; ***p < 0.001 versus mock transfected (N = 3). (C) Etoposide treatment increased levels of miR-711 and Ang-1 within the RISC. Neurons were collected 6 h after etoposide treatment, subjected to RIP with Ago2 antibodies, and samples used for qPCR analysis. qPCR quantification of miR-711 and Ang-1 levels in precipitates after RIP. Data represent the mean ± SEM. Student's t-test; ***p < 0.001 versus untreated RCNs. (D) miR-711 targets 3′-UTRs of Ang-1 mRNA. RCNs were transfected with either control vector (pmirGLO) or pmir-Ang-1 reporter plasmids and co-transfected with either negative control miR (−ve) or miR-711 mimic. Luciferase activity was analyzed 24 h after transfection. Normalized luciferase activities are shown as the percentage relative to cells transfected with plasmid alone, which was set as 100%. Experiments were performed in triplicate. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; ***p < 0.001 versus RCN co-transfected with pmir-Ang-1 reporter plasmid and negative control miR (−ve) mimic (N = 3). (E) Ang-1 does not attenuate etoposide-induced miR-711 upregulation. Neurons were treated with etoposide alone or with etoposide and recombinant Ang-1 for 24 h. Level of miR-711 was quantified by qPCR. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; **p < 0.01 versus cnt. (F) Central administration of miR-711 hairpin inhibitor after TBI increased the level of Ang-1. Whole-tissue lysates from mouse cortex 24 h after TBI and i.c.v. administration of miR-711 or −ve Con hairpin inhibitors were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against Ang-1 and β-actin. Protein levels of were quantified by densitometry, normalized to β-actin, and presented as fold change compared to noninjured controls. Data represent the mean ± SEM. One-way ANOVA. SNK post-hoc test, ***p < 0.001 versus noninjured; ⁺p < 0.05 vs. −ve Con miR inhibitor CCI group (N = 6). Ago2, argonaute 2, RISC catalytic component; Ang-1, angiopoietin-1; ANOVA, analysis of variance; i.c.v., intracerebroventricular; miR, microRNA; mRNA, messengter RNA; qPCR, quantitative real-time polymerase chain reaction; RCN, rat cortical neuron; RIP, RNA-binding protein immunoprecipitation; RISC, RNA-induced silencing complex; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; SNK, Student–Newman–Keuls; TBI, traumatic brain injury; 3′-UTRs, 3′ untranslated regions.