Transient focal ischemia induces extensive temporal changes in rat cerebral microRNAome

Abstract

MicroRNAs (miRNAs) are approximately 22 nucleotides long, noncoding RNAs that control cellular function by either degrading mRNAs or arresting their translation. To understand their functional significance in ischemic pathophysiology, we profiled miRNAs in adult rat brain as a function of reperfusion time after transient middle cerebral artery occlusion. Of the 238 miRNAs evaluated, 8 showed increased and 12 showed decreased expression at least at 4 out of 5 reperfusion time points studied between 3 h and 3 days compared with sham. Of those, 17 showed >5 fold change. Bioinformatics analysis indicated a correlation between miRNAs altered to several mRNAs known to mediate inflammation, transcription, neuroprotection, receptors function, and ionic homeostasis. Antagomir-mediated prevention of mir-145 expression led to an increased protein expression of its downstream target superoxide dismutase-2 in the postischemic brain. In silico analysis showed sequence complementarity of eight miRNAs induced after focal ischemia to 877 promoters indicating the possibility of noncoding RNA-induced activation of gene expression. The mRNA expression of the RNases Drosha and Dicer, cofactor Pasha, and the pre-miRNA transporter exportin-5, which modulate miRNA biogenesis, were not altered after transient middle cerebral artery occlusion. Thus, the present studies indicate a critical role of miRNAs in controlling mRNA transcription and translation in the postischemic brain.

Figure 1

(A) Correlation of the hybridization signal intensities of all the expressed miRNAs between two sham samples showed very few differences (top panel). Whereas, correlation of a sham sample with a 24 h MCAO sample showed several differentially expressed miRNAs (middle panel). The number of miRNAs altered progressed temporally from 3 h to 3 days of reperfusion after transient focal ischemia (bottom panel). (B) Hierarchical cluster analysis of the miRNAs altered after transient MCAO. The color code in the heat maps is linear with green as the lowest and red as the highest. The miRNAs that were increased in expression were shown in green to red, whereas the miRNAs that were decreased in expression were shown from red to green. The figure shows the data from 36 individual microarrays (six arrays/group). The individual expression signal of each miRNA in each array was clustered using Euclidean distance function. The dendrograms (tree diagrams) show the grouping of miRNAs according to the order in which they were joined during the clustering. The miRNAs with most similar expression patters were placed next to each other.

Figure 2

In situ hybridization showed very little staining for rno-mir-145 in the cerebral cortex of sham (A and a). In a representative rat subjected to transient MCAO and 1 day of reperfusion, the contralateral cortex (B) and periinfarct cortex (C) showed little staining, but the ischemic core in the ipsilateral cortex (D and d) showed increased hybridization signal for rno-mir-145. However, rno-mir-137 showed a uniform staining density in the sham cortex (E and e), contralateral cortex (F), periinfarct cortex (G), and decreased staining in the core of the ipsilateral cortex (H and h) of a rat subjected to transient MCAO and 1 day of reperfusion. Scale bar is 50 µm.

Figure 3

Real-time PCR analysis confirmed the changes in the levels of seven miRNAs at 1 day of reperfusion after transient MCAO observed by microarray analysis (A). The expression levels of none of the miRNA synthetic machinery proteins (Drosha, Pasha, Dicer, and Exportin-5) studied using real-time PCR changed compared with sham control at either 3 h or 1 day of reperfusion after transient MCAO (B). Bars represent mean ± s.d. of n = 4/group.

Figure 4

Antagomir-145 infusion resulted in increased SOD2 protein expression and decreased area of infarction at 1 day of reperfusion. Panels A–C show representative Cresyl violet-stained sections from sham (A), control antagomir/MCAO (B), and antagomir-145/MCAO (C) groups. The black lines on the images B and C separates the infarct from the relatively normal periinfarct brain tissue. Panels a, b, and c show SOD2 immunoreactive neurons in sham (a), control antagomir/MCAO (b), and antagomir-145/MCAO (c) groups in the boxed areas indicated in the A, B, and C, respectively. The boxed areas in a, b, and c was shown in a higher magnification in a^|, b^|, and c^|, respectively. Similar staining pattern was observed in four rats per group. Panel D is a western blot showing SOD2 and HSP70 protein levels in the control antagomir (C1, C2, C3, and C4) and antagomir-145 (A1, A2, A3, and A4) treated rats subjected to transient MCAO and 1 day of reperfusion. Panel E shows the rat miR-145 sequence with a complementary 8-bp targeting site in the 3′-UTR of rat SOD2 mRNA (GenBank# NM_017051). Real-time PCR analysis showed that antagomir-145 treatment significantly prevented the postischemic mir-145 induction compared with control antagomir treatment (E). Antagomir-145 treatment had no effect on the postischemic expression of four other miRNAs (F). P < 0.05 compared with control antagomir group (by Student t-test).