Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA

Fuminori Sato, Masatoshi Kurokawa, Nobuhiko Yamauchi, Masa Aki Hattori

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)C538-C545
JournalAmerican Journal of Physiology - Cell Physiology
Volume291
Issue number3
DOIs
Publication statusPublished - Sep 13 2006

Fingerprint

Myostatin
Myoblasts
Gene Silencing
Small Interfering RNA
Chickens
Genes
Skeletal Muscle Fibers
Myogenin
Muscle Development
Cell Fusion
RNA Interference
Luciferases
Muscle
Cell Differentiation
Assays
Skeletal Muscle
Fusion reactions

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cell Biology

Cite this

Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA. / Sato, Fuminori; Kurokawa, Masatoshi; Yamauchi, Nobuhiko; Hattori, Masa Aki.

In: American Journal of Physiology - Cell Physiology, Vol. 291, No. 3, 13.09.2006, p. C538-C545.

Research output: Contribution to journalArticle

@article{f505a746e52a4b90aad9c91440f651b7,
title = "Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA",
abstract = "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.",
author = "Fuminori Sato and Masatoshi Kurokawa and Nobuhiko Yamauchi and Hattori, {Masa Aki}",
year = "2006",
month = "9",
day = "13",
doi = "10.1152/ajpcell.00543.2005",
language = "English",
volume = "291",
pages = "C538--C545",
journal = "American Journal of Physiology",
issn = "0363-6143",
publisher = "American Physiological Society",
number = "3",

}

TY - JOUR

T1 - Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA

AU - Sato, Fuminori

AU - Kurokawa, Masatoshi

AU - Yamauchi, Nobuhiko

AU - Hattori, Masa Aki

PY - 2006/9/13

Y1 - 2006/9/13

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=33748414066&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33748414066&partnerID=8YFLogxK

U2 - 10.1152/ajpcell.00543.2005

DO - 10.1152/ajpcell.00543.2005

M3 - Article

C2 - 16611734

AN - SCOPUS:33748414066

VL - 291

SP - C538-C545

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6143

IS - 3

ER -