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Abstract
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.
Highlights
MicroRNAs are 17 to 23 nucleotides long, evolutionarily conserved non-coding RNAs that control protein translation by binding to the complementary seed sequences in the 39UTRs of mRNAs [1]
Restoring miR-29c levels protects against OGD, while providing excess miR29c had no effect under normal conditions
Knocking-down miR-29c promotes cell death under normal conditions. These observations show that miR-29c is a prosurvival miRNA under physiological conditions and its down-regulation after ischemia is a promoter of cell death
Summary
MicroRNAs (miRNAs) are 17 to 23 nucleotides long, evolutionarily conserved non-coding RNAs that control protein translation by binding to the complementary seed sequences in the 39UTRs of mRNAs [1]. We and others reported that ischemia significantly alters cerebral miRNAome and specific miRNAs modulate either neuroprotection or neuronal death in the postischemic brain [2,3,4,5,6,7,8,9,10,11]. Our studies showed that miR-29c is one of the highly expressed miRNAs in rat brain that was down-regulated in a sustained manner during the acute phase (2 h to 3 days) after focal ischemia [3]. We currently evaluated the functional significance of miR-29c in ischemic cell death under in vitro (oxygen-glucose deprivation; OGD in PC12 cells) and in vivo (transient middle cerebral artery occlusion; MCAO in adult rats) conditions. We further evaluated if repressor element 1 silencing transcription factor (REST) aka neuron-restrictive silencing factor (NRSF) controls miR-29c expression
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