ISSN 1214-0287 (on-line), ISSN 1214-021X (printed)
J Appl Biomed
Volume 10 (2012), No 2, p 63-70
DOI 10.2478/v10136-011-0023-2

Effect of shRNA mediated Smad4 gene silencing on the fibrosis of C2C12 myoblasts

Shiqiu Chen, Jiwu Chen, Shiyi Chen, Hongyun Li, Jia Jiang

Address: Jiwu Chen, Department of Sports Medicine, Huashan Hospital, Fudan University Shanghai Medical College, Shanghai 200040, China
chen6786789@sina.com

Received 29th September 2011.
Revised 1st November 2011.
Published online 12th December 2011.

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SUMMARY
Our present study aimed to investigate the effect of lentiviral-mediated RNAi using short hairpin RNA (shRNA) targeting Smad4 on TGF-beta1 induced fibrosis. shRNAs targeting Smad4 were designed and the most efficient shRNA was screened. This shRNA was introduced into a lentiviral vector which was used to infect C2C12 myoblasts, and then the Smad4 expression was detected. Cells were divided into: C2C12 cells group, TGF-beta1 induction group, transfection group, and transfection after TGF-beta1 induction group. C2C12 myoblasts were transfected with lentivirus carrying Smad4-shRNA and treated with TGF-beta1 to induce the differentiation into myofibroblasts. Fluorescence Real-time-PCR and the western blot assay were employed to detect the expressions of collagen I and alpha-SMA. The results showed that the protein and mRNA expression of Smad4 in the C2C12 cells transfected with Smad4-shRNA1 was significantly reduced when compared with C2C12 before transfection. In the TGF-beta1 induction group, the mRNA expressions of alpha-SMA and collagen I were significantly increased as compared to the C2C12 cells group. In the transfection after TGF-beta1 induction group, the mRNA expressions of alpha-SMA and collagen I were significantly increased compared to the transfection group, and the protein expressions significantly increased, respectively. In the transfection after TGF-beta1 induction group, the mRNA expressions of alpha-SMA and collagen I were significantly decreased compared to the TGF-beta1 induction group, and the protein expressions significantly reduced, respectively. The results indicate that suppression of Smad4 expression can efficiently inhibit the TGF-beta1 induced fibrosis in myoblasts. The findings suggest Smad4 may become a novel target for the treatment of skeletal muscle fibrosis.

KEY WORDS
skeletal muscle injury; fibrosis; transforming growth factor-beta1; C2C12 myoblast

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