title: TGF-β1 stimulates human Tenon's capsule fibroblast proliferation by miR-200b and its targeting of p27/kip1 and RND3.
abstract: OBJECTIVE
  To evaluate the role of miR-200b expression in the proliferation of human Tenon's capsule fibroblasts (HTFs) induced by transforming growth factor-beta 1 (TGF-β1).
  
  
  METHODS
  Human Tenon's capsule fibroblasts were treated with various doses of TGF-β1 for 24 hours. Cell proliferation was quantified by the cell counting kit-8 (cck-8) assay, cell cycle analysis, 5-ethynyl-2-deoxyuridine (EdU) assay, and analysis of cyclin E, cyclin D1, and proliferating cell nuclear antigen (PCNA) expression. MicroRNA-200b (miR-200b) was detected by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), and its potential target genes were validated by the luciferase assay and Western blot analysis. The effect of miR-200b on the proliferation of HTFs was analyzed using both miR200b-mimic and inhibitor-transfected HTFs and confirmed in p27/kip1 and RND3 (the target of miR-200b) knockdown cells.
  
  
  RESULTS
  The proliferation of the TGF-β1-treated HTFs increased significantly in a dose- and time-dependent manner. Treatment with 5 ng/mL TGF-β1 caused an upregulation of miR-200b. The luciferase assay identified p27/kip1 and RND3 as target genes for miRNA-200b, which was confirmed by the expression of p27/kip1 and RND3 and their downstream products (cyclinE and cyclinD1) in the TGF-β1-treated cells. Transforming growth factor-β1 and miR-200b mimics enhanced the proliferation of HTFs; suppressed the expression of p27/kip1 and RND3; and subsequently stimulated the expression of cyclinE, cyclinD1, and PCNA. The miR-200b inhibitor attenuated the effects of TGF-β1 on HTFs. Furthermore, knockdown of p27/kip1 and RND3 resulted in an increase in cell proliferation and expression of the proliferation-related genes.
  
  
  CONCLUSIONS
  MicroRNA-200b acts as a stimulant for the proliferation of HTFs by targeting p27/kip1 and RND3.
authors.affiliation: Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Tong
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Fu
authors.forename: Xinyu
authors.initials: X
authors.lastname: Xu
authors.forename: Shuxin
authors.initials: S
authors.lastname: Fan
authors.forename: Hong
authors.initials: H
authors.lastname: Sun
authors.forename: Ya
authors.initials: Y
authors.lastname: Liang
authors.forename: Kai
authors.initials: K
authors.lastname: Xu
authors.forename: Zhilan
authors.initials: Z
authors.lastname: Yuan
authors.forename: Yingbin
authors.initials: Y
authors.lastname: Ge
mesh_terms: D015153:Blotting, Western; D002452:Cell Count; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000096:biosynthesis / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D005901:Glaucoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D058475:Tenon Capsule / Q000378:metabolism / Q000473:pathology*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D020741:rho GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C436291:CDKN1B protein, human; C533896:MIRN200 microRNA, human; D035683:MicroRNAs; D053773:Transforming Growth Factor beta1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D012313:RNA; C508705:RND3 protein, human; D020741:rho GTP-Binding Proteins
keywords: TGF-β1; cell proliferation; glaucoma; human Tenon's capsule fibroblasts; microRNA-200b