上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

miRNA138体外抑制C2C12细胞纤维化研究

薛鸣丰，龚遂良，戴加平，陈 刚，胡隽宇   

  1. 嘉兴市第二医院骨科，  嘉兴 314000
  • 出版日期:2015-01-28 发布日期:2015-01-29
  • 作者简介:薛鸣丰（1978—）， 男， 副主任医师， 学士; 电子信箱: xuemfeng@163.com。
  • 基金资助:

    嘉兴市科技局科技计划项目（2012AY1071-7）

Study on inhibition of fibrosis of C2C12 cells in vitro by miRNA138

XUE Ming-feng, GONG Sui-liang, Dai Jia-ping, CHEN Gang, HU Jun-yu   

  1. Department of Orthopaedic Surgery, Jiaxing Second Hospital, Jiaxing 314000, China
  • Online:2015-01-28 Published:2015-01-29
  • Supported by:

    Science and Technology Project of Jiaxing Technology Bureau, 2012AY1071-7

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摘要：

目的 探讨转化生长因子-β1 （TGF-β1）诱导小鼠成肌细胞C2C12建立体外纤维化模型，观察miRNA138含量改变对细胞纤维化指标的影响，分析miRNA138对细胞纤维化的影响是否通过其靶基因Smad4实现。方法 设计并合成miRNA138-mimics、miRNA138-inhibitor、miRNA138-NC，经脂质体转染至C2C12细胞，转染后细胞再经TGF-β1 （10 ng/mL）诱导48 h；Real-time PCR检测胞内miRNA138表达，Western blotting检测胞内纤维化相关蛋白Smad4、vimentin、α-SMA及collagenⅠ表达变化，确认纤维化模型是否成功建立并分析胞内miRNA138表达改变对细胞纤维化指标的影响；CCK-8法检测miRNA138表达改变对纤维化过程中C2C12细胞增殖活性的影响；生物信息学分析miRNA138与Smad4理论结合位点并通过荧光素酶报告基因实验进行验证；构建Smad4基因表达载体，并与miRNA138-mimics共转染C2C12细胞，转染后细胞再经TGF-β1诱导发生纤维化，Western blotting检测细胞纤维化相关蛋白变化，分析外源Smad4表达对于miRNA138抑制细胞纤维化作用的影响。结果 miRNA138-mimics转染C2C12细胞后细胞内miRNA138表达明显上升；miRNA138-inhibitor转染后，C2C12细胞内miRNA138表达下降；转染对照组细胞内miRNA138表达无明显变化；蛋白检测结果显示：TGF-β1诱导C2C12细胞48 h，胞内Smad4、vimentin、α-SMA和collagenⅠ蛋白表达均明显升高，细胞对数生长期增殖活性明显增强，表明细胞纤维化模型成功建立；细胞内miRNA138过表达明显抑制了C2C12细胞纤维化；荧光素酶活性检测数据显示，miRNA138与Smad4的预测靶位是客观存在的；在C2C12细胞内高表达外源Smad4基因，可明显减弱miRNA138-mimics对细胞纤维化相关蛋白的抑制作用。结论 miRNA138可以抑制TGF-β1诱导的C2C12细胞纤维化，是通过抑制其靶蛋白Smad4表达实现的。

关键词: Smad4, 纤维化, miRNA138, C2C12

Abstract:

Objective To explore the establishment of a fibrosis model in vitro by inducing rat myoblast C2C12 by transforming growth factor-β (TGF-β), observe the effects of variations of the miRNA138 level on cell fibrosis indexes, and analyze whether the effects of miRNA138 on cell fibrosis was achieved by its target gene Smad4. Methods The miRNA138-mimics, miRNA138-inhibitor, and miRNA138-NC were designed, synthesized and transfected into C2C12 cells by lipofectminae. Transfected cells were then induced by TGF-β1 of 10 ng/mL for 48 h. The expression of miRNA138 was detected by Real-time PCR. The variations of expressions of fibrosis related proteins Smad4, vimentin, α-SMA, and collagen I were detected by the Western blotting to verify whether the fibrosis model had been successfully established and analyze the effects of variations of the miRNA 138 expression on cell fibrosis indexes. The effects of variations of the miRNA 138 expression on the proliferation activity of C2C12 during the course of fibrosis were measured by the CCK-8. Bioinformatic software was used to analyze the theoretical binding site of miRNA138 and Smad4, which was verified by the luciferase reporter gene test. The Smad4 gene expression vector was constructed and co-transfected into C2C12 cells with miRNA-mimics. Transfected cells were induced by TGF-β1 for fibrosis. The variations of fibrosis related proteins were detected by the Western blotting and the effects of expression of exogenous Smad4 on the inhibition of cell fibrosis by miRNA138 were analyzed. Results The expression of miRNA138 significantly increased after C2C12 cells were transfected by miRNA138-mimics and decreased after C2C12 cells were transfected by miRNA138-inhibitor. The expression of miRNA138 of the control group had no significant changes. Results of protein detection showed that expressions of Smad4, vimentin, α-SMA, and collagen I were significantly increased and the cell proliferation activity in logarithmic phase was enhanced after C2C12 cells were induced by TGF-β1 for 48 h, which indicated that the cell fibrosis model was successfully established. The over-expression of miRNA138 significantly inhibited the fibrosis of C2C12 cells. The data of luciferase activity detections showed that the predicted binding site of miRNA138 and Smad4 existed. High expression of exogenous Smad4 in C2C12 cells significantly impaired the inhibition of cell fibrosis by miRNA138-mimics. Conclusion MiRNA138 can inhibit the fibrosis of C2C12 cells induced by TGF-β1 via suppressing the expression of its target protein Smad4.

Key words: Smad4, fibrosis, miRNA138, C2C12