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.
- MeSH
- barvení a značení metody využití MeSH
- fibróza genetika komplikace terapie MeSH
- financování organizované MeSH
- fluorescenční mikroskopie využití MeSH
- kosterní svaly cytologie zranění MeSH
- lidé MeSH
- malá interferující RNA genetika metabolismus MeSH
- myoblasty kosterní cytologie fyziologie patologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody využití MeSH
- protein Smad4 genetika metabolismus MeSH
- satelitní buňky kosterního svalu fyziologie metabolismus MeSH
- statistika jako téma MeSH
- transformující růstový faktor beta1 genetika metabolismus MeSH
- western blotting využití MeSH
- Check Tag
- lidé MeSH
