title: Recombinant adenoviral microRNA-206 induces myogenesis in C2C12 cells.
abstract: BACKGROUND
  The expression of microRNA-206 (miR-206) is high in skeletal muscle but low in most other tissues. The expression of miR-206 is increased in muscular dystrophy, suggesting its involvement in the pathogenesis of muscle diseases. To determine the role of miR-206 in muscle cell differentiation and explore a possible gene therapy vector, we constructed a miR-206 adenoviral expression vector (AdvmiR-206) and tested for transfection into C2C12 stem cells.
  
  
  METHODS
  A 355-bp PCR amplicon from C57B6 mouse skeletal muscle genomic DNA was inserted into the adenoviral shuttle vector pAdTrack-CMV, which was then co-transformed with the adenoviral backbone plasmid pAdEasy-1 into competent E. coli BJ5183 bacteria. The specificity and function of this recombinant adenoviral MiR-206 were studied in C2C12 cells by Northern blot, immunofluorescence, Western blot, and flow cytometry.
  
  
  RESULTS
  Increased expression of miR-206 in AdvmiR-206 transfected C2C12 cells (P < 0.001) and resulted in morphological and biochemical changes over time that were similar to serum deprivation, including elongated cells and increased myosin heavy chain proteins. Even in the absence of serum deprivation, miR-206 overexpression accounted for a 50% reduction of S-phase cells (P < 0.01). Moreover, in untransfected C2C12 cells, the introduction of miR-206-specific antisense oligoribonucleotides inhibited the normal response to serum deprivation. Twenty-four hours after lipofection of antisense oligoribonucleotides, the number of elongated cells was reduced by half (P < 0.01).
  
  
  CONCLUSIONS
  Collectively, these data support a role for miR-206 in myoblast differentiation. We foresee potential applications for the AdvmiR-206 vector in research and therapy.
authors: State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, 3rd Military Medical University, Chongqing, PR China.
authors: Weiwei
authors: W
authors: Zhang
authors: Tao
authors: T
authors: Wang
authors: Yongping
authors: Y
authors: Su
authors: Wang
authors: W
authors: Li
authors: Lynn T
authors: LT
authors: Frame
authors: Guoping
authors: G
authors: Ai
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002453:Cell Cycle; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D005786:Gene Expression Regulation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics*; D024510:Muscle Development / Q000235:genetics*; D018482:Muscle, Skeletal; D032446:Myoblasts / Q000378:metabolism* / Q000821:virology*; D009928:Organ Specificity / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D014162:Transfection
chemical_list: D035683:MicroRNAs; C540669:Mirn206 microRNA, mouse