Suppression of type I collagen expression by miR-29b via PI3K, Akt, and Sp1 pathway in human Tenon's fibroblasts.
OBJECTIVE
To evaluate the expression profile of microRNAs (miRNAs) and their roles in human Tenon's fibroblasts (HTFs), and to establish an miRNA-based gene-silencing method for antifibrosis in vitro.


METHODS
The miRNA expression profile was analyzed by microarray using quiescent and transforming growth factor beta 1 (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. 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 analysis.


RESULTS
In all, 38 miRNAs were identified to be upregulated and 31 downregulated, in TGFβ1-stimulated HTFs. Among those, the miR-29b, downregulated 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. 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.
Department of Ophthalmology, Second Xiangya Hospital, Central South University, Changsha, Hunan Province, People's Republic of China.
Ning
N
Li
Juanlian
J
Cui
Xuanchu
X
Duan
Huihui
H
Chen
Fang
F
Fan
D015153:Blotting, Western; D024042:Collagen Type I / Q000378:metabolism*; D000090602:Collagen Type I, alpha 1 Chain; D015536:Down-Regulation; D005347:Fibroblasts / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D035683:MicroRNAs / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D058475:Tenon Capsule / Q000378:metabolism*; D015854:Up-Regulation
D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D035683:MicroRNAs; D019869:Phosphatidylinositol 3-Kinases; D011494:Protein Kinases; D051057:Proto-Oncogene Proteins c-akt; C532045:MIRN29a microRNA, human; C065999:Sp1 kinase