マイオスタチンに対するRNA干渉法による骨格筋形成の調節
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Skeletal muscle is one of the most important morpho-functional organs, and its atrophy causes severe conditions such as muscular dystrophies. In the craniofacial region, skeletal deformities and malocclusion are often caused by hyper-and hypotrophy of masticatory muscles or the tongue. Recently, RNA interference (RNAi)-based gene therapy has been expected as a safe remedy without side effects ; therefore, we used RNAi to control the expression of myostatin, a negative regulator of skeletal muscle formation, for the purpose of developing a new method to regulate skeletal muscle volume in this study. We constructed a plasmid vector containing the full-length of myostatin cDNA (pcDNA3.1-Mst-myc) and a vector for silencing mRNA for either myostatin (pSi-Mst) or green fluorescence protein (GFP) sequence as a negative control (pSi-N). When these vectors were transiently co-transfected in COS-1 cells, the myostatin expression level was notedly reduced. For better understanding of the effect of gene silencing by pSi-Mst on mouse myoblastic cells, we generated stable transfectants overexpressing pcDNA3.1-Mst-myc in mouse myoblast cell line C2C12. The proliferation ability in C2C12 cells overexpressing myostatin was accelerated by transfection with pSi-Mst, but not with pSi-N. Furtheremore, the myogenic differentiation of C2C12 cells overexpressing myostatin, transfected with pSi-Mst, showed a higher expression of MyoD and myogenin than control cells transfected with pSi-N. In addition, they exhibited slight acceleration of myotube formation and a slight increase in myosin heavy chain (MHC) expression. Finally, to investigate the effect of synthetic 19 nucleotide-long double-stranded RNA (Mst-siRNA) in vivo, we injected Mst-siRNA/atelocollagen complex into the masseter and biceps femoris muscles in four mice. After two weeks, myostatin protein levels in those muscles were significantly decreased, but not in the contralateral muscles. Moreover, dramatically increased muscle mass and myofibril size were observed following myostatin down-regulation. These results suggest that it may be possible to regulate skeletal muscle volume and to develop a new treatment for various muscle disorders by RNAi technique.
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