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Abstract #56952 Published in IGR 16-2
TGF-β1 stimulates human Tenon's capsule fibroblast proliferation by miR-200b and its targeting of p27/kip1 and RND3
Tong J; Fu Y; Xu X; Fan S; Sun H; Liang Y; Xu K; Yuan Z; Ge YInvestigative Ophthalmology and Visual Science 2014; 55: 2747-2756
PURPOSE: To evaluate the role of miR-200b expression in the proliferation of human Tenon's capsule fibroblasts (HTFs) induced by transforming growth factor-beta 1 (TGF-β1). METHODS: Human Tenon's capsule fibroblasts were treated with various doses of TGF-β1 for 24 hours. Cell proliferation was quantified by the cell counting kit-8 (cck-8) assay, cell cycle analysis, 5-ethynyl-2-deoxyuridine (EdU) assay, and analysis of cyclin E, cyclin D1, and proliferating cell nuclear antigen (PCNA) expression. MicroRNA-200b (miR-200b) was detected by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and its potential target genes were validated by the luciferase assay and Western blot analysis. The effect of miR-200b on the proliferation of HTFs was analyzed using both miR200b-mimic and inhibitor-transfected HTFs and confirmed in p27/kip1 and RND3 (the target of miR-200b) knockdown cells. RESULTS: The proliferation of the TGF-β1-treated HTFs increased significantly in a dose- and time-dependent manner. Treatment with 5 ng/mL TGF-β1 caused an upregulation of miR-200b. The luciferase assay identified p27/kip1 and RND3 as target genes for miRNA-200b, which was confirmed by the expression of p27/kip1 and RND3 and their downstream products (cyclinE and cyclinD1) in the TGF-β1-treated cells. Transforming growth factor-β1 and miR-200b mimics enhanced the proliferation of HTFs; suppressed the expression of p27/kip1 and RND3; and subsequently stimulated the expression of cyclinE, cyclinD1, and PCNA. The miR-200b inhibitor attenuated the effects of TGF-β1 on HTFs. Furthermore, knockdown of p27/kip1 and RND3 resulted in an increase in cell proliferation and expression of the proliferation-related genes. CONCLUSIONS: MicroRNA-200b acts as a stimulant for the proliferation of HTFs by targeting p27/kip1 and RND3.
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Classification:
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
3.6 Cellular biology (Part of: 3 Laboratory methods)
2.3 Sclera (Part of: 2 Anatomical structures in glaucoma)
12.8.10 Woundhealing antifibrosis (Part of: 12 Surgical treatment > 12.8 Filtering surgery)
