Abstract
Purpose: :
To evaluate the expression profile of microRNAs (miRNAs) and their roles in human tenon’s fibroblasts (HTFs), and to establish a miRNA-based gene silencing method for antifibrosis in vitro.
Methods: :
The miRNA expression profile was analyzed by microarray using quiescent and TGFβ1-stimulated primary HTFs, respectively. Candidate miRNAs were identified by quantitative RT-PCR. miRNAs potentially targeting fibrosis-related genes were predicted using a published algorithm (TargetScan; Envisioneering Medical Technologies, St. Louis, MO). Predicted fibrosis-related genes regulated by candidate miRNAs were confirmed by transfection of the miRNA into HTF culture (with or without TGFβ1 treatment), followed by quantitative RT-PCR and Western Blot.
Results: :
Total of 38 miRNAs were identified to be upregulated, and 31 down-regulated, in TGFβ1-stimulated HTFs. Among those, the miR-29b, down-regulated in TGFβ1-treated HTFs, targeted a cadre of mRNAs that encode proteins involved in fibrosis, including PI3Kp85α, Sp1, and collagen type I alpha1 (Col1A1). Treatment of HTFs with TGFβ1 activated the PI3K-Akt-Sp1 pathway and, consequently, induced an increase in the expression of type I collagen. Down-regulation of miR-29b by introducing an antisense miRNA into cultured HTFs partly induced the expression of PI3Kp85α, Akt, Sp1 and Col1A1, whereas overexpression of miR-29b inhibited the PI3K-Akt-Sp1 pathway and attenuated the expression of Col1A1.
Conclusions: :
miR-29b acted as a suppressor of type I collagen gene by repressing the PI3K/Akt/Sp1 pathway in HTFs. Overexpression 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 attenuating scar formation after glaucoma filtering surgery.
Keywords: signal transduction • wound healing • gene microarray