| Central nervous system injruy leeds severe functional dysfunction, which threats human health and affects quality of life. To date there is no effective treatment for CNS injury due to the failure of axonal regeneration after injury. Following CNS injury, there are small molecules in the which can inhibit axon regeneration, including Nogo-A, MAG(myelin-associated glycoprotein) and CSPG(Chondroitin sulphate proteoglycans). These axonal inhibitors bind with Nogo receptor on cell membrane and activates Rho followed by ROCK activation, which leads to alteration of cytoskeleton in neurons and growth cone collapse, retraction.MicroRNA is A class of small, non-coding RNA, which can bind to the complementary site in the 3’ UTR of mRNA of target gene, then regulating gene expression at the level of translation. Recently, accumulated studies demonstrated that miRNA played an important role in the axon growth, branching, guidance and regeneration, e.g., miR-34 regulated the neurite outgrowth and spinal morphology,, miR-124 regulated neurite branching, and lin-4 miRNA was involved in axon guidance.MiR-133 b is a commonly dys-regulated miRNA in numerous forms of cancer, including prostate cancer, lung cancer and gastrointestinal cancer. In CNS, miR-133 b is enriched in the midbrain and plays an important role in differentiation and degeneration of midbrain dopaminergic neurons through repressing Pitx3. Moreover, recent studies showed that miR-133 b was implicated in the functional recovery of spinal cord injury and stroke. However, the cellular role and possible mechanism of miR-133 b in neurite outgrowth of neural cells remained unclear. In addition, it is also unclear whether miR-133 b is involved in the axonal regeneration after CNS injury.Objective: To explore the role of miR-133 b in axonal growth of neural cells and possible mechanism.Methods:PC12 cells and primary cortical neurons(PCNs) were transfected with lenti-miR-133 b, lenti-miR-133 b inhibitor, plasmid-shRNA-RhoA, plasmid-RhoA and their negative controls. PC12 cells were transfected with plasmids by using Lipofectamine 2000 following the manufacturer’s instructions. For lentivirus infections, 2×104 PC12 cells or 1×105 PCNs were exposed to 1×106 virus particles for 12 hours. In some experiments, the cells were also cultured with LY294002(10uM, an inhibitor of PI3K), or PD098059(10uM, an inhibitor of MAP kinase(MAPK) kinase, MEK1). After 48 hours of transfection, the levels of proteins and mRNA or mi RNA were evaluated by Western blotting and qRT-PCR, respectively. Moreover, the neurite outgrowth was analyzed by Image J. For neurite inhibition experiments, PCNs were plated in 24-well plates coated with poly-l-lysine or CSPG substrates. Cells were then transfected with lenti-miR-133b/lenti-miR-NC or treated with 50 uM Y27632. In addition, proliferation and apoptosis of PC12 cells was assessed by the MTT assay and TUNEL(terminal deoxynucleotidyl transferasemediated biotinylated UTP nick end labeling) staining, respectively. Results:Part 1: In PC12 cells, miR-133 b regulates the axon outgrowth. Over expression of miR-133 b promotes the axon growth, conversely, down regulation of miR-133 b expression in PC12 cells inhibits the neurite outgrowth. MiR-133 b dose not have any effect on neural differentiation, apoptosis or proliferation in PC12 cells.Part 2: Over-expression of miR-133 b inhibits the expression of RhoA protein, whereas did not influence the mRNA expression. Down-regulation the RhoA protein expression by using shRNA can mimic the effect of miR-133 b on promoting axon outgrowth. In addition, overexpression of RhoA could attenuate the neurite growth effects of miR-133 b.Part 3. We observed that miR-133 b activated the phosphorylation of ERK1/2 and Akt(Ser 473), but have no effect on p38 phosphorylation. Moreover, PD98059 and LY294002(inhibitors of MEK1 and PI3 K, respectively) strongly inhibited neurite growth induced by miR-133 b in PC12 cells. Finally, knockdown of RhoA expression enhanced phosphorylation of Erk1/2 and Akt, similar to that induced by miR-133 b.Part 4. The results showed that overexpression of miR-133 b decreased the protein levels of RhoA in PCNs, but had no effect on the expression of RhoA mRNA. Moreover, 3 days after transfection, we observed that miR-133 b enriched PCNs extended longer axons than PCNs transfected with miR-NC. miR- 133 b reversed the inhibitory effect of CSPG on neurite extension similar to Y27632(an inhibitor of ROCK).Conclusion: Taken together, our observations suggest that mi R-133 b promotes neuritic outgrowth by suppression of RhoA, thereby activating MEK/ERK and PI3K/Akt pathways. Additionally, overexpression of miR-133 b can overcome axon growth restrictions from CSPG through inhibition of RhoA/ROCK pathway... |
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