Background: Neurogenesis is associated with functional recovery after stroke. However, the underlying molecular mechanisms have not been fully investigated. We hypothesized that stroke changes miRNA profiles in neural progenitor cells (NPCs), which is closely associated with stroke-induced neurogenesis.
Methods and Results: Using a novel cross-linking immunoprecipitation to crosslink Ago2-RNA complexes followed by RNA sequencing (Ago2 CLIP-seq) approach, we profiled the entire small RNA population in NPCs harvested from the non-ischemic and ischemic subventricular zone (SVZ) of ischemic animals (n=6/group). Ago2 CLIP-seq analysis showed that over 7 million reads were found in normal and ischemic NPC libraries. In non-ischemic NPCs, the top 10 highly enriched miRNAs were miR-26, -27b, -127, -99b, -191, -125b-5p, let-7f, let-7i, -541 and -151. In contrast, stroke significantly changed the top 10 enriched miRNAs. Bioinformatics analysis revealed that these miRNAs could target genes involved in cytoskeleton remodeling, WNT, TGF signaling and cell cycle. Moreover, we detected 18 novel miRNAs (>50 reads) in the non-ischemic and ischemic NPCs. Pc-17172 is one of the novel miRNAs specifically down-regulated in NPCs, which was further validated by quantitative RT-PCR. Conditional knockout of Dicer reduced its expression, indicating that novel pc-17172 follows canonical miRNA biogenesis pathway. To test its biological function, we knocked down its expression by locked nucleic acid and found that the % of Tuj1+ neuroblasts was significantly increased (10 ± 1% vs 6 ± 1% in control group, p<0.05, n=3), whereas the % of BrdU+ cells was decreased (44 ± 5% vs 84 ± 4% in control group, p<0.05). Meanwhile, over-expression of pc-17172 by transfection of its mimics into NPCs significantly decreased the % of Tuj1+ neuroblasts (7 ± 1% vs 12 ± 2% in control group, p<0.05) and increased the % of BrdU+ cells (93 ± 2% vs 80 ± 3%, p<0.05), suggesting reduced pc-17172 is involved in stroke-induced neurogenesis.
Conclusion: Collectively, the present study reveals profiles of miRNomes in non-ischemic and ischemic NPCs, which provide molecular basis to further investigate the role of miRNAs in mediating adult neurogenesis under physiological and pathophysiological conditions.