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