MiR-133b Promotes neurite outgrowth by targeting RhoA expression.

BACKGROUND: MicroRNA-133b (miR-133b) has been shown to play a critical role in spinal cord regeneration. The aim of this study was to investigate the cellular role of miR-133b in neural cells. METHODS: PC12 cells and primary cortical neurons (PCNs) were transfected with lenti-miR-133b, lenti-miR-133b inhibitor, plasmid-shRNA-RhoA, plasmid-RhoA and their negative controls. After 48 hours of transfection, the levels of proteins and mRNA or miRNA were evaluated by Western blotting and qRT-PCR, respectively. Moreover, the neurite outgrowth was analyzed by Image J. For pharmacological experiments, inhibitors of MEK1/2 kinase (PD98059), phosphoinositide-3 kinase (PI3K) (LY294002) and ROCK (Y27632) were added into the culture medium. RESULTS: Overexpression of miR-133b in PC12 cells enhanced neurite outgrowth. Conversely, inhibition of miR-133b reduced neurite length. We further identified RhoA as a target and mediator of mir-133b for neurite extension by Western blot and knockdown experiment. Moreover, overexpression of RhoA could attenuate the neurite growth effects of miR-133b. Also, we observed that miR-133b activated MEK/ERK and PI3K/Akt signaling pathway by targeting RhoA. Finally, in PCNs, miR-133b also increased axon growth and attenuated axon growth restrictions from chondroitin sulfate proteoglycans (CSPG). CONCLUSIONS: In summary, our study suggested that miR-133b regulated neurite outgrowth via ERK1/2 and PI3K/Akt signaling pathway by RhoA suppression.

Neurogenin 2 converts mesenchymal stem cells into a neural precursor fate and improves functional recovery after experimental stroke.

BACKGROUND: Neurogenin2 (Ngn2) is a proneural gene that directs neuronal differentiation of progenitor cells during development. Here, we investigated whether Ngn2 can reprogram MSCs to adopt a neural precursor fate and enhance the therapeutic effects of MSCs after experimental stroke. METHODS: In vitro, MSCs were transfected with lenti-GFP or lenti-Ngn2. Following neuronal induction, cells were identified by immunocytochemistry, Western blot and electrophysiological analyses. In a stroke model induced by transient right middle cerebral artery occlusion (MCAO), PBS, GFP-MSCs or Ngn2-MSCs were injected 1 day after MCAO. Behavioral tests, neurological and immunohistochemical assessments were performed. RESULTS: In vitro, Ngn2-MSCs expressed neural stem cells markers (Pax6 and nestin) and lost the potential to differentiate into mesodermal cell types. Following neural induction, Ngn2-MSCs expressed higher levels of neuron-specific proteins MAP2, Tuj1 and NeuN, and also expressed voltage-gated Na+ channel, which was absent in GFP-MSCs. In vivo, after transplantation, Ngn2-MSCs significantly reduced apoptotic cells, decreased infarct volume, and increased the expression of VEGF and BDNF. Finally, Ngn2-MSCs treated animals showed the highest functional recovery among the three groups. CONCLUSIONS: Ngn2 was sufficient to convert MSCs into a neural precursor fate and transplantation of Ngn2-MSCs was advantageous for the treatment of stroke rats.