Suppression Of Type Ⅰ Collagen Expression By MiR-29b Via PI3K, Akt, And Sp1Pathway In Human Tenon's Fibroblasts

BACKGROUNDGlaucoma is the first irreversible ocular disease caused blinding in the world. In the drug and laser can not control the intraocular pressure (IOP), filtering surgery (Glaucoma Filtration Surgery, GFS) is still the main means for treatment of glaucoma. However, the Tenon's capsule fibroblasts hyperplasia and scar formation leads to the high failure of filtering operation after1year up to15%. Bleb scarring is the main reason leading to operation failure. The TGF-beta induced Tenon's capsule fibroblasts (HTFs) to the myofibroblast transformation, its persistence and the synthesis of collagen and other extracellular matrix is the central link of filtering bleb scarring.Over the years, many domestic and foreign experts committed to research to prevent postoperative scar formation. Because the application time of antiscaring drugs often are not obvious acted or due to the time of action is too long, wide effect on ocular tissues causes a series of complications. Therefore, elucidating the pathogenesis postoperative scarring and actively explore more specific treatment are necessary.miRNA is a recently discovered family of non-protein-coding micromolecule RNA with19～25nucleotides long. They regulate gene expression by either degradation or translational repression of a target mRNA through combination with non-coding region at3'terminal. miRNA is involved not only in the regulation of cell proliferation, apoptosis and differentiation, but also in the individual development and metabolism. miRNA has shown great potential in heart, liver, kidneys and other body organs in the treatment of fibrotic diseases and has become a new target for drug of the antifibrosis and angiogenesis for further development.Bleb scarring has similar treatment challenges to organ fibrosis. Detection the same miRNA expression means that common antifibrotic strategies might exist. Successful miRNA therapy, which has been used for other organ fibrosis might be possible for the prevention of post-operation scarring.PURPOSETo study the correlation between microRNAs (miRNA) and post-operation scarring. To evaluate the expression profile of miNAs and their roles in human tenon's fibroblasts (HTFs), and to establish a miRNA-based gene silencing method for antifibrosis in vitro.METHODSHuman subconjunctival fibroblasts (HTFs) were obtained from excised Tenon's capsule specimens during strabismus surgery. We cultured primary HTFs, used TGFβ1inducing HTFs and underwent immunohistochemistry identification. 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 TGFβ1treatment). By quantitative RT-PCR and Western blot, we studied the mechanism of reaction between miR-29b and PI3K/Akt/Spl pathway, and attempted to clarify the relationship between miRNA and post-operation scar formation,RESULTSTotal of38miRNAs were identified to be upregulated, and31down-regulated, in TGF(31-stimulated HTFs. Among those, the miR-29b, down-regulated in TGF(31-treated HTFs, targeted a cadre of mRNAs that encode proteins involved in fibrosis, including PI3Kp85a, Spl, and collagen type Ⅰ alphal (CollA1). Treatment of HTFs with TGFβ1activated the PI3K/Akt/Spl pathway and, consequently, induced an increase in the expression of type Ⅰ collagen. Overexpression of miR-29b inhibited the PI3K/Akt/Sp1pathway and attenuated the expression of CollA1.CONCLUSIONS The activated HTFs induced by TGF-β1were the successful cell model of post-surgery scar formation. The HTFs miRNA expression differences existed with or without induction by TGF-β1. Among them, miR-29b targeted regulation of PI3Kp85a, Spl and CollAl. miR-29b acted as a suppressor of type Ⅰ collagen gene by repressing the PI3K/Akt/Spl 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.