Altered expression of miRNA-21 and its targets in the hippocampus after traumatic brain injury

Abstract

Traumatic brain injury (TBI) initiates many different signaling cascades throughout the brain that impact both pathophysiological and neuroprotective processes. Cellular mechanisms that can modulate these processes may play an important role in determining the nature and extent of the damage suffered after TBI and therefore influence overall outcome after injury. MicroRNAs (miRNAs) are an important class of noncoding regulatory RNAs providing an epigenetic mechanism for the regulation of protein expression levels of target genes. We report that miR-21 expression is significantly up-regulated in the hippocampus after rodent TBI, with expression levels peaking by 3 days postinjury and returning to near sham levels by 15 days postinjury. In situ localization of miR-21 transcripts indicates widespread expression in normal brain, with a pronounced increase in expression after TBI evident throughout the cortex and hippocampus, including the dentate gyrus and CA3 cell layer. We used a combination of the miRanda, TargetScan, and PicTar prediction algorithms to identify 99 potential target genes that possess miR-21 binding sites within their 3' untranslated regions. Analysis of these genes' annotated Gene Ontology molecular function and biological process terms revealed an overrepresentation of genes involved in enzyme-linked receptor signaling, transcriptional regulation, and developmental processes. These results suggest that increased miR-21 expression in the hippocampus may influence multiple components of TBI pathophysiology.

Copyright © 2010 Wiley-Liss, Inc.

Figures

Figure 1.

MicroRNA-21 expression is altered in the hippocampus after TBI. A) Summary of miR-21 expression altered after TBI as detected by miRNA microarray analysis (Redell et al., 2008a). B) Changes in miR-21 expression level detected by qRT-PCR analysis of total RNA isolated from ipsilateral hippocampal tissue harvested at the indicated times after injury. C) qRT-PCR analysis of changes in miR-21 expression in the contralateral hippocampus at the indicated times after injury. Data is presented as the mean±SD of the difference in threshold cycle as compared to sham animals (ΔCt = CtSham - CtTBI). Group sizes for sham, 3hr, 24hr, and 3d post-TBI were n=6, 5, 6, and 6, respectively. Asterisks (*) indicate p≤0.01 determined by 1-way ANOVA or Kruskal-Walis ANOVA on ranks; SD= standard deviation; CV= coefficient of variation.

Figure 2.

Increased miR-21 expression is observed in cells throughout the brain after injury. Representative in situ images taken from a sham (A-G) and a 3d post-TBI (H-N) brain showing increased miR-21 expression after injury. A scrambled miRNA probe with no similarity to known miRNAs was used to assess the level of non-specific hybridization in sham (A) and 3d post-TBI (H) brain tissue. Injury did not substantially alter background hybridization signal. Hybridization using a miR-21 antisense probe revealed increased signal intensity at 3d post-injury (I) relative to sham (B) throughout the hippocampus, including in the dentate gyrus, and CA1-CA3 subfields, as well as in cells located outside of the neuronal cell layers. The boxed regions of B and I are shown at higher magnification in the following panels: CA1 (C,J); CA3 (D, K); dentate gyrus (E, L); hippocampal fissure (F, M); cortex (G, N). Scale bars in A, B, H and I represent 500 μm; scale bars in C-G and J-N represent 50 μm. Arrowheads indicate presumed vessels in the hippocampal fissure. DG, dentate gyrus; CA1-CA3, Cornu Ammonis 1–3; HF, hippocampal fissure.

Figure 3.

Western blots for the miR-21 predicted targets Tiam1, peli-1, and PDCD4. Total hippocampal extract (8 μg) was separated by SDS-PAGE and the resulting blot probed with antibodies directed against (A) PDCD4, (B) Tiam1, and (C) pellino-1, Images of the immunoreactive bands are shown above their respective bars graphs. Data are presented as mean±SEM of percent signal intensity, with sham signal intensity set to 100%. N=4/group; Asterisks (*) indicate p≤0.01 by 1-way ANOVA followed by post-hoc comparisons.

Figure 4.

Overrepresented gene ontologies within the predicted miR-21 target genes. (A) Graphical representation of the relationships of the overrepresented molecular function terms, (B) Graphical representation of the relationships of the overrepresented biological process terms, (C) Relative representation of each GO term within the population group versus the study group, and the associated calculated p-values. Data was compared using Bonferroni’s method with a conservative correction for multiple comparisons. An adjusted p-value <0.05 was considered significant. B-biological process; M-molecular function.