Abstract 3139: The microRNA 17-92 Cluster Mediates Sonic Hedgehog-driven Neurogenesis In Ischemic Neural Progenitor Cells

Abstract

Background: The Sonic hedgehog (Shh) pathway regulates stroke-induced neurogenesis. The present study investigated the functional role of the microRNA 17-92 (miR17-92) cluster in this process. Methods and Results: Analysis of miRNA microarray and real-time RT-PCR revealed that stroke substantially increased levels of individual members of the miR17-92 cluster: miR-18a (1.8±0.3), miR-19a (2.5±0.4), and miR-92a (1.9±0.3) expression in neural progenitor cells (NPCs) harvested from the subventricular zone (SVZ) of ischemic rats (n=6). Overexpression of the miR17-92 cluster in cultured NPCs significantly increased NPC proliferation measured by the number of BrdU positive cells (52±4% vs 28±2% in empty vector, n=3/group, p<0.05). Concurrently, overexpression of the miR17-92 cluster reduced PTEN (phosphatase and tensin homolog), a target of the miR17-92 cluster, protein levels by 70% compared to levels in NPCs transfected with an empty vector. PTEN suppresses cell proliferation. These data suggest that the stroke-upregulated miR17-92 cluster enhances NPC proliferation via downregulation of PTEN. To examine whether Shh regulates miR17-92 cluster expression, NPCs were incubated with recombinant human Shh (rhShh, 100ng/ml). We found that rhShh significantly (p<0.05) increased levels of individual members of the miR17-92 cluster: miR-18a (2.1±0.1), miR-19a (1.3±0.7), miR-19b (1.5±0.6) and miR-92a (1.9±0.8). Blockage of a Shh receptor Smo with cyclopamine suppressed rhShh-increased levels of miR-18a (0.9±0.08), miR-19a (0.7±0.01), miR-19b (0.6±0.05) and miR-92a (0.8±0.04). Attenuation of endogenous Shh in NPCs with siRNA also substantially decreased levels of miR-18a (0.6±0.1), miR-19a (0.4±0.05) and miR-92a (0.6±0.1) compared with levels in NPCs transfected with scrambled siRNA (1.0±0.2, n=3), indicating that Shh regulates miR17-92 expression. MYC is a downstream target of Shh. Western blots showed that stroke increased the protein level of N-MYC 1.8 fold in SVZ tissues and incubation of NPCs with rhShh elevated N-MYC levels by 1.8 fold, which was abrogated by cyclopamine (1.3 fold). N-MYC transduction resulted in significant increases in expression of the primary miR17-92 cluster (2.1±0.3 vs 1.0±0.2 in control group, n=3, p<0.05). These data suggest that the Shh pathway recruits N-MYC to regulate miR17-92 cluster expression in NPCs. Conclusion: Our data suggest a functional role of the miR17-92 cluster in mediating stroke-induced neurogenesis by the SHH/MYC signaling pathway, which provides new insight into molecular mechanisms of stroke-induced neurogenesis.