title: Inhibition of Nogo expression to promote repair after spinal cord injury.
abstract: BACKGROUND
  One of the reasons for poor neuroregeneration after central nervous system injury is the presence of inhibitory factors such as Nogo. Here, we tested the inhibition of Nogo by RNA interference both in vitro and in vivo, using recombinant adenovirus-mediated transfection of short hairpin RNAs, to explore a new method of treatment for spinal cord injury.
  
  
  METHODS
  We designed and cloned two Nogo-specific short hairpin RNAs and an unrelated short hairpin RNA, packaged the clones into adenovirus, and amplified the recombinant virus in 293 cells. We then tested the inhibition of Nogo expression both in vitro in adenovirus-transfected oligodendrocytes and in vivo in spinal cord tissue from adenovirus-transfected spinal cord injury model rats. We tested Nogo expression at the mRNA level by reverse-transcription PCR and at the protein level by Western blotting and immunohistochemistry.
  
  
  RESULTS
  In vitro, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 51% and 49%, respectively, compared with Nogo expression in cells transfected with the unrelated control small hairpin RNA (P < 0.005). Similarly, Nogo protein expression decreased by 50% and 48%, respectively (P < 0.005). In vivo, in spinal cord injury model rats, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 45% and 40%, respectively, compared with Nogo expression in spinal cord injury model rats transfected with the unrelated control short hairpin RNA (P < 0.005). The Nogo protein level was similarly decreased.
  
  
  CONCLUSIONS
  We were successful in specifically downregulating Nogo at the mRNA and protein levels by adenovirus-mediated delivery of short hairpin RNAs, both in vitro and in vivo. This confirms the effectiveness of RNA interference for the inhibition of Nogo gene expression and the efficiency of using adenovirus for delivery. Thus gene therapy may be an effective treatment for spinal cord injury.
authors.affiliation: Department of Orthopedics, the Second Hospital, Jilin University, Changchun, Jinlin, China.
authors.forename: Hong-hui
authors.initials: HH
authors.lastname: Sun
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.forename: Hai-tao
authors.initials: HT
authors.lastname: Yu
authors.forename: Ning
authors.initials: N
authors.lastname: Kong
authors.forename: Guo-min
authors.initials: GM
authors.lastname: Liu
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D006801:Humans; D007150:Immunohistochemistry; D009185:Myelin Proteins / Q000235:genetics / Q000378:metabolism*; D000070798:Nogo Proteins; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013119:Spinal Cord Injuries / Q000628:therapy*
chemical_list: D009185:Myelin Proteins; D000070798:Nogo Proteins; D034741:RNA, Small Interfering; C000603199:RTN4 protein, human; C000603197:Rtn4 protein, rat