MicroRNAs (miRNAs) are ~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.

Background and Purpose Long noncoding RNAs (lncRNAs) play a significant role in cellular physiology. We evaluated the effect of focal ischemia on the expression of 8,314 lncRNAs in rat cerebral cortex using microarrays. Methods Ischemia was induced by transient middle cerebral artery occlusion. Genomic and transcriptomic correlates of the stroke-responsive lncRNAs and the transcription factor binding sties in their promoters were evaluated with bioinformatics. Results 359 lncRNAs were upregulated (>2-fold) and 84 were downregulated (<0.5 fold) at 3h to 12h of reperfusion following MCAO compared to sham. 62 stroke-responsive lncRNAs showed >90% sequence homology with exons of protein-coding genes. Promoters of stroke-responsive lncRNA genes and their homologous protein-coding genes showed highly overlapping transcription factor binding sites. Despite presence of ORFs, lncRNAs did not form any product when subjected to in vitro translation. Conclusions Stroke significantly alters cerebral lncRNA expression profiles.

Recent studies showed that stroke extensively alters cerebral microRNA (miRNA) expression profiles and several miRNAs play a role in mediating ischemic pathophysiology. We currently evaluated the significance of miR-29c, a highly expressed miRNA in rodent brain that was significantly down-regulated after focal ischemia in adult rats as well as after oxygen-glucose deprivation in PC12 cells. Bioinformatics indicated that DNA methyltransferase 3a (DNMT3a) is a major target of miR-29c and co-transfection with premiR-29c prevented DNMT3a 3′UTR vector expression. In PC12 cells, treatment with premiR-29c prevented OGD-induced cell death (by 58±6%; p<0.05). Furthermore, treatment with antagomiR-29c resulted in a 46±5% cell death in PC12 cells. When rats were treated with premiR-29c and subjected to transient focal ischemia, post-ischemic miR-29c levels were restored and the infarct volume decreased significantly (by 34±6%; p<0.05) compared to control premiR treated group. DNMT3a siRNA treatment also significantly curtailed the post-OGD cell death in PC12 cells (by 54±6%; p<0.05) and decreased the post-ischemic infarct volume in rats (by 30±5%; p<0.05) compared to respective control siRNA treated groups. The miR-29c gene promoter showed specific binding sites for the transcription factor REST and the miR-29c promoter vector expression was curtailed when cotransfected with a REST expressing plasmid. Furthermore, treatment with REST siRNA prevented the post-ischemic miR-29c down-regulation and DNMT3a induction in PC12 cells and curtailed ischemic cell death (by 64±9%; p<0.05) compared to control siRNA treatment. These studies suggest that miR-29c is a pro-survival miRNA and its down-regulation is a promoter of ischemic brain damage by acting through its target DNMT3a. Furthermore, REST is an upstream transcriptional controller of miR-29c and curtailing REST induction prevents miR-29c down-regulation and ischemic neuronal death.