Effect of shRNA mediated Smad4 gene silencing on the fibrosis of C2C12 myoblasts
Effect of shRNA mediated Smad4 gene silencing on the fibrosis of C2C12 myoblasts
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shRNA介导的Smad4基因沉默对C2C12成肌细胞纤维化的影响
DOI：
10.2478/v10136-011-0023-2
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发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Jiang, Jia
中科院分区：
文献类型：
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作者：
Chen, Shiqiu;Chen, Jiwu;Jiang, Jia
本研究的目的是探讨慢病毒介导的RNAi对TGF-β 1诱导的纤维化的影响。设计靶向Smad 4的shRNA并筛选最有效的shRNA。将该shRNA导入慢病毒载体，感染C2 C12成肌细胞，检测Smad 4的表达。将细胞分为C2 C12细胞组、TGF-β 1诱导组、转染组和TGF-β 1诱导后转染组。用携带Smad 4-shRNA的慢病毒转染C2 C12成肌细胞，并用TGF-β 1处理以诱导分化成肌成纤维细胞。采用实时荧光定量PCR和western blot检测I型胶原和α-SMA的表达。结果显示，转染Smad 4-shRNA 1的C2 C12细胞中Smad 4蛋白和mRNA的表达均较转染前明显降低。与C2 C12细胞组相比，TGF-β 1诱导组的α-SMA和I型胶原mRNA表达显著增加。TGF-β 1诱导后转染组，α-SMA和I型胶原的mRNA表达较转染组显著增加，蛋白表达也较转染组显著增加。TGF-β 1诱导后转染组中α-SMA和I型胶原的mRNA表达较TGF-β 1诱导组明显降低，蛋白表达也明显降低。结果表明，抑制Smad 4的表达可以有效地抑制TGF-β 1诱导的成肌细胞纤维化。研究结果表明Smad 4可能成为治疗骨骼肌纤维化的新靶点。
Our present study aimed to investigate the effect of lentiviral-mediated RNAi using shRNA targeting Smad4 on TGF-beta 1 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-beta 1 induction group, transfection group, and transfection after TGF-beta 1 induction group. C2C12 myoblasts were transfected with lentivirus carrying Smad4-shRNA and treated with TGF-beta 1 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-beta 1 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-beta 1 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-beta 1 induction group, the mRNA expressions of alpha-SMA and collagen I were significantly decreased compared to the TGF-beta 1 induction group, and the protein expressions significantly reduced, respectively. The results indicate that suppression of Smad4 expression can efficiently inhibit the TGF-beta 1 induced fibrosis in myoblasts. The findings suggest Smad4 may become a novel target for the treatment of skeletal muscle fibrosis.