Abstract 2726: The Mir-17-92 Microrna Cluster Enhances Axonal Outgrowth In A Rat Cortical Neurons After Oxygen-glucose Deprivation
Background: There is a paucity of studies on the biological function of MicroRNAs (miRNAs) in axons. We investigated the role of the miR-17-92 cluster, one of the first oncogenic miRNAs, on axonal outgrowth in cortical neurons subjected to oxygen-glucose deprivation (OGD).
Methods: Cortical neurons harvested from embryonic day-17 Wistar rats were cultured in a Standard Neuron Device, and were subjected to OGD for 3h. Quantitative real-time RT-PCR (qRT-PCR) was used to selectively analyze the abundance of individual members of the miR-17-29 cluster in axons and somas. Cortical neurons were transfected with a pCAG-vector carrying a rat miR-17-92 cluster, or miRNA hairpin inhibitors against rat miR-18a and miR-19a. Axonal outgrowth was monitored with time-lapse microscopy. Western blot was performed to analyze phosphatase tensin homolog deleted on chromosome 10 (PTEN) and the mammalian target of rapamycin (mTOR).
Results: qRT-PCR revealed that compared to somas, axons had relatively abundant levels of six individual members of the miR-17-92 cluster: miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92. The presence of miR-18a and miR-19a in axons was confirmed by in situ hybridization. OGD challenge (n=5) upregulated axonal levels of the six individual members of the miR-17-92 cluster by approximately two fold compared to axonal levels without OGD (n=5, p<0.05). Concurrently, OGD increased the number of axons (213 ± 27, n=5) compared to the number of axons without OGD (175 ± 22, n=5, P<0.05). Overexpression of the miR-17-92 cluster further increased the number of OGD-augmented axons (222 ± 16 vs 179 ± 9 in the empty vector, n=4, P<0.05). Overexpression of the miR-17-92 cluster in OGD challenged neurons significantly increased the length of axons (1.0 ± 0.1 vs 0.4 ± 0.1, μ m/5min in the empty vector, n=3/group, P<0.01), whereas attenuation of the endogenous miR-19a suppressed OGD-induced axonal elongation (0.7 ± 0.1, μ m/5min, n= 3, P<0.05) compared with the length of axons in neurons transfected with cel-miR-67 (1.2 ± 0.2, n=3). Western blot analysis revealed that overexpression of the miR-17-92 cluster reduced miR-19 targeting protein PTEN by 87% and activated mTOR by 194% (n= 5, P<0.05).
Conclusion: The present study provides the first evidence that cortical neurons contain the miR-17-92 cluster in axons and that the miR-17-92 cluster mediates axonal outgrowth in neurons subjected to OGD likely through targeting PTEN. These data suggest that the miR-17-92 cluster may play a role in axonal remodeling after stroke.
- © 2012 by American Heart Association, Inc.