title: MiR-133b Promotes neurite outgrowth by targeting RhoA expression.
abstract: 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.
authors.affiliation: Department of Neurosurgery, First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
authors.forename: Xiao Cheng
authors.initials: XC
authors.lastname: Lu
authors.forename: Jin Yu
authors.initials: JY
authors.lastname: Zheng
authors.forename: Lin Jun
authors.initials: LJ
authors.lastname: Tang
authors.forename: Bao Sheng
authors.initials: BS
authors.lastname: Huang
authors.forename: Kai
authors.initials: K
authors.lastname: Li
authors.forename: Yi
authors.initials: Y
authors.lastname: Tao
authors.forename: Wan
authors.initials: W
authors.lastname: Yu
authors.forename: Rong Lan
authors.initials: RL
authors.lastname: Zhu
authors.forename: Shuai
authors.initials: S
authors.lastname: Li
authors.forename: Li Xin
authors.initials: LX
authors.lastname: Li
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D002867:Chromones / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D002867:Chromones; D005419:Flavonoids; D035683:MicroRNAs; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D009025:Morpholines; D016376:Oligonucleotides, Antisense; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D034741:RNA, Small Interfering; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; C549446:MIRN133 microRNA, rat; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C578418:ROCK1 protein, rat; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one