ROLE OF Smad4 ON FIBROSIS OF TENDON DERIVED FIBROBLASTS INDUCED BY TRANSFORMING GROWTH FACTOR β1 BY TARGETED REGULATION OF miRNA219-5P
10.7507/1002-1892.20160128
- Author:
Hongjiang RUAN
1
Author Information
1. Department of Orthopaedics, Sixth Affiliated People's Hospital, Shanghai Jiaotong University School of Medicine
- Publication Type:Journal Article
- Keywords:
Fibrosis;
miRNA219-5P;
Smad4;
Tendon adhesion;
Tendon derived fibroblasts
- From:
Chinese Journal of Reparative and Reconstructive Surgery
2016;30(5):641-646
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE: To investigate the effect of Smad4 on the fibrosis of tendon derived fibroblasts (TDFs) induced by transforming growth factor β1(TGF-β1) by targeted regulation of miRNA219-5P (miR219-5P). METHODS: The tendons donated by the volunteers were harvested to isolate and culture TDFs. The 3rd generation cells were used for experiment. Chemically synthesized miR219-5P mimics, miR219-5P inhibitor, and negative control sequences were transfected into TDFs. The gene expression of miR219-5P in TDFs was detected by real-time PCR, and the protein expression of Smad4 in TDFs was detected by Western blot at 48 hours after transfection. The combining sites of miR219-5P and Smad4 in 3'UTR district were predicted by informatics software. Wild type and mutant type reporter gene expression vectors were constructed and then targeted verification was carried out by the luciferase reporter gene test. Transfected TDFs were then induced by TGF-β1. The proliferation activity of the cells were measured by the cell counting kit 8 after culturing for 24, 48, and 72 hours. The expressions of fibrosis related proteins in TDFs were detected by Western blot at 72 hours. RESULTS: After TDFs were transfected by miR219-5P mimics, miR219-5P expression was significantly up-regulated, but the expressions of Smad4 was decreased subsequently (P<0.05). Intracellular expression of miR219-5P was inhibited by miR219-5P mimics inhibitor, however, the protein expression of Smad4 was significantly increased (P<0.05). Luciferase reporter gene test showed that luciferase activities were significantly decreased in pGL3-WT-Smad4+mimics group, but were significantly increased in pGL3-WT-Smad4+inhibitor group when compared with pGL3-WT-Smad4 transfected group (P<0.05), but no significant difference was found between GL3-MT-Smad4+mimics and pGL3-MT-Smad4+inhibitor groups (P>0.05). Cell proliferation and the fibrosis related proteins were increased in TGF-β1 induced TDFs, however, decreased in TGF-β1 induced TDFs after transfected by miR219-5P inhibitor (P<0.01). CONCLUSIONS: miR219-5P can significantly inhibit fibrosis of TDFs induced by TGF-β1 by down-regulating Smad4 expression.
