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A retrovirus‐based system to stably silence GDF‐8 expression and enhance myogenic differentiation in human rhabdomyosarcoma cells
Author(s) -
Yang Zhuo,
Zhang Jingwu,
Cong Haolong,
Huang Zenghong,
Sun Lunquan,
Liu Changmei,
Tien Po
Publication year - 2008
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1216
Subject(s) - retrovirus , biology , rna interference , myogenesis , myostatin , microbiology and biotechnology , c2c12 , cell cycle , propidium iodide , cell growth , cellular differentiation , cell , cell culture , apoptosis , myocyte , rna , programmed cell death , genetics , gene
Background Myostatin, also called GDF‐8, a secreted growth and differentiating factor that belongs to the transforming growth factor‐β superfamily, is a known negative regulator of myogenesis in vivo . Overexpression of GDF‐8 contributes to the lack of differentiation in human rhabdomyosarcoma (RMS) cells. We investigated whether a retrovirus‐based RNA interference (RNAi) system against GDF‐8 expression in human RMS cells would enhance myogenic differentiation. Methods A retrovirus‐based RNAi system was developed that utilized the U6‐RNA polymerase III promoter to drive efficient expression and deliver the GDF8‐specific short hairpin RNAs (shRNAs) in human RMS cell A204. In this system, the retrovirus vector was integrated into the host cell genome and allowed stable expression of shRNAs. GDF‐8 expression was determined by real‐time polymerase chain reaction and western blotting analysis. An 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay was performed to determine the cell proliferation. Myogenic differentiation markers were monitored by western blotting analysis. Cell cycle and apoptosis was determined by propidium iodide staining and analysed in a flow cytometer. Results In the siGDF8 A204 cell pools, the levels of both GDF‐8 mRNA and protein were dramatically reduced by this RNAi system. In differentiation conditions, inhibition of myostatin synthesis led to enhanced cell cycle withdrawal, consequently stimulated myogenic differentiation and increased the rate of tumor cell apoptosis. Conclusions The results demonstrate that deactivation of myostatin by using retrovirus‐based RNAi thus may be useful for therapy in rhabdomyosarcomas. Copyright © 2008 John Wiley & Sons, Ltd.