A retrovirus-based system to stably silence GDF-8 expression and enhance myogenic differentiation in human rhabdomyosarcoma cells.
BACKGROUND
Myostatin, also called GDF-8, a secreted growth and differentiating factor that belongs to the transforming growth factor-beta 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.
Molecular Virology Research Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China.
Zhuo
Z
Yang
Jingwu
J
Zhang
Haolong
H
Cong
Zenghong
Z
Huang
Lunquan
L
Sun
Changmei
C
Liu
Po
P
Tien
D017209:Apoptosis / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D055435:Myostatin; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D012208:Rhabdomyosarcoma / Q000235:genetics*; D014161:Transduction, Genetic; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
C525911:MSTN protein, human; D055435:Myostatin; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta