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Jun Tong, Yuxuan Fu, Xinyu Xu, Shuxin Fan, Hong Sun, Ya Liang, Kai Xu, Zhilan Yuan, Yingbin Ge; TGF-β1 Stimulates Human Tenon's Capsule Fibroblast Proliferation by miR-200b and its Targeting of p27/kip1 and RND3. Invest. Ophthalmol. Vis. Sci. 2014;55(4):2747-2756. doi: https://doi.org/10.1167/iovs.13-13422.
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© ARVO (1962-2015); The Authors (2016-present)
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).
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.
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.
MicroRNA-200b acts as a stimulant for the proliferation of HTFs by targeting p27/kip1 and RND3.
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