Weiwei Zhang, Tao Wang, Yongping Su, Wang Li, Lynn T. Frame, Guoping Ai
Med Sci Monit 2011; 17(12): BR364-371
Available online: 2011-12-01
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.
Material/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.
Keywords: Muscle Development - genetics, Muscle, Skeletal, MicroRNAs - genetics, Mice, Inbred C57BL, Mice, Gene Expression Regulation, Cell Line, Cell Differentiation - genetics, Cell Cycle, Animals, Adenoviridae - genetics, Myoblasts - virology, Organ Specificity - genetics, Recombination, Genetic - genetics, Transfection