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Ning Li, Xuanchu Duan; Suppression of Collagen Production by miR-29b Via Phosphatidylinositol 3-kinase, Akt, and Sp1 in Human Tenon's Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2011;52(14):620.
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© ARVO (1962-2015); The Authors (2016-present)
To reveal the microRNA (miRNA) expression profile and related roles in human tenon’s fibroblasts (HTFs), and to establish an activated-HTFs-specific gene silencing method for antifibrosis in vitro.
miRNA expression profiling was analyzed in quiescent and in TGFβ1-activated primary HTFs by microarray and then confirmed the candidate miRNA, such as miR-29b, by quantitative PCR. miRNAs potentially targeting important fibrosis-related genes, as defined by expression pattern and implication in disease, were predicted using a published algorithm (TargetScan; Envisioneering Medical Technologies, St. Louis, MO). The predicted targets for repression by miR-29b were substantiated by cotransfection of miRNA mimic and luciferase reporter plasmids in cell culture. The activity of miR-29b against fibrotic target genes was analyzed by quantitative PCR and Western blot analysis of HTFs (with or without TGFβ1) that were transiently transfected with miR-29b mimic and inhibitor or were treated with LY294002, a specific inhibitor of PI3K.
The upregulated and downregulated miRNAs in activated HTFs were 38 miRNAs and 31 miRNAs, respectively. The miR-29b, down-regulated in the HTFs after exposure to TGFβ1, targeted a cadre of mRNAs that encoded proteins involved in fibrosis, including phosphatidylinositol 3-Kinase (PI3K), Sp1, collagen type I, alpha1 (Col1A1) and collagen type III, alpha 1 (Col3A1). Treatment of HTFs with TGFβ1 caused both an activation of the PI3K, Akt, Sp1 and an increase in expression of multiple collagens. Down-regulation of miR-29b with anti-miRs in vitro induced the expressions of PI3K, Akt, Sp1, Col1A1 and Col3A1, whereas over-expression of miR-29b in fibroblasts reduced the fibrotic response as LY294002. The miR-29b-induced inhibition rate of TGFβ1 reached 60% and above.
miR-29b acted as a supressor of collagen gene expression via the sequential actions of PI3K, Akt and Sp1 in HTFs. Upregulation of miR-29b protected subconjunctival tissues against collagen production and fibrosis. These findings provided a novel rationale for the development of miRNA-based strategies for the attenuation of scar formation after glaucoma filtering surgery.
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