Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA.
Myostatin (GDF-8) is known to negatively regulate skeletal muscle mass in myogenesis, but few studies have been conducted on the function of endogenous GDF-8 in primary myoblasts. The present study was performed to assess the function of GDF-8 by RNA interference using primary culture of chicken embryonic myoblasts in which myoblasts were differentiated into myotubes. An active form of small interfering RNA (siRNA-1) targeting GDF-8 mRNA was introduced into myoblasts, and an inactive form of siRNA (siRNA-2) was used as a negative control. GDF-8 transcript level was significantly reduced 24 h after the introduction of siRNA-1 to 25% of the control, whereas a 52-kDa GDF-8 precursor was reduced to 45% of the control at 48 h. However, siRNA-2 did not decrease GDF-8 transcript level. When GDF-8-mediated promoter activity was measured chronologically by means of a pGL(CAGA)(10)-constructed luciferase reporter assay, a concomitant change in activity was initiated after 24 h. The activity rapidly decreased 30 h after siRNA-1 introduction, whereas high activity was maintained at 30-42 h in the control and siRNA-2-treated myoblasts. Myogenic factors such as MyoD and p21, but not myogenin, were altered after 72 h. Cell fusion of the multinucleated myotubes was delayed by the siRNA-1 introduction, and myotubes with aggregated nuclei were shorter and wider. These results strongly suggest that deficiency of GDF-8 delays cell differentiation and causes great alterations in the cellular morphology of chicken embryonic myotubes.
Sato
Fuminori
F
Laboratory of Reproductive Physiology and Biotechnology, Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Graduate School Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Kurokawa
Masatoshi
M
Yamauchi
Nobuhiko
N
Hattori
Masa-Aki
MA
D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D048430:Cell Shape; D002642:Chick Embryo; D016895:Culture Media, Serum-Free; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D017570:MyoD Protein / Q000235:genetics; D032446:Myoblasts / Q000166:cytology*; D018008:Myogenin / Q000235:genetics; D055435:Myostatin; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000502:physiology*
D016895:Culture Media, Serum-Free; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017570:MyoD Protein; D018008:Myogenin; D055435:Myostatin; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta