Background: To determine the involvement of MicroRNA-200b (miR-200b) in human Tenon's fibroblasts (HTFs) fibrosis.
Methods: HTFs were treated with 10 ng/ml TGF-β1 for 24 h. Expression of α-smooth muscle actin (α-SMA), Fibronectin and Collagen1A1 (COL1A1) was assessed by western blot and immunofluorescence. MiR-200b was detected by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and its potential target genes were validated by luciferase assay. The effects of miR-200b and PTEN (the target gene) on HTFs fibrogenesis were evaluated using miR-200b mimics or inhibitor transfected HTFs, and confirmed with PTEN-specific small interfering RNA (siRNA) and PTEN inhibitor.
Results: TGF-β1 up-regulated miR-200b and promoted the expression of α-SMA, Fibronectin and COL1A1 in HTFs, and the effects could be strengthened or hampered by adding miR-200b mimics or inhibitor, respectively. Luciferase assay identified PTEN as the target gene of miR-200b, and decreased PTEN level due to up-regulation of miR-200b was found to correlate with intensified expression of α-SMA, Fibronectin and COL1A1. Determination of PTEN's role in fibrosis was carried out by adding PTEN specific siRNA or PTEN inhibitor into HTFs, which led to intensified expression of fibrosis-related proteins.
Conclusion: TGF-β1-mediated miR-200b induces HTFs fibrosis through suppressing PTEN signaling pathway.
Keywords: TGF-β1; fibrosis; glaucoma; human Tenon’s fibroblasts; miR-200b.