title: MicroRNA-29b Overexpression Decreases Extracellular Matrix mRNA and Protein Production in Human Corneal Endothelial Cells.
abstract: OBJECTIVE
  MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. We reported that levels of microRNA (miR)-29 family are decreased in corneas of patients with Fuchs endothelial corneal dystrophy (FECD). The miR-29 family regulates the production of extracellular matrix (ECM) proteins. Accumulation of ECM proteins in Descemet membrane is an important pathologic change in FECD. In this study, we transfected miR-29b into human corneal endothelial cells and tissues and evaluated ECM protein expression levels.
  
  
  METHODS
  An immortalized Fuchs human corneal endothelial cell line (iFECD) was established by infection of corneal endothelial cells from patients with FECD with hTERT lentivirus. MiR-29b was transfected into iFECD, and the expression levels of ECMs collagen type 1 alpha 1 (COL1A1), collagen type 4 alpha 1 (COL4A1), and laminin gamma 1 (LAMC1) were evaluated with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot. Expression level of LAMC1 protein in miR-29b-transfected donor corneal endothelium was also evaluated by Western blot.
  
  
  RESULTS
  Compared with control, miR-29b expression level after transfection of iFECD was increased to 335.6% (±91.0%), and ECM expression levels were significantly decreased. Compared with control, qRT-PCR demonstrated reduction of ECM to the following levels: COL1A1: 1.9% (±0.4%); COL4A1: 7.1% (±1.7%); and LAMC1: 21.5% (±2.7%). Western blot showed reduced protein expression: COL1A1: 4.8% (±3.2%); COL4A1: 42.5% (±25.0%); and LAMC1: 44.8% (±3.1%). In miR-29b-transfected corneal tissue, LAMC1 protein expression level was decreased to 14.4% (±20.5%).
  
  
  CONCLUSIONS
  Overexpression of miR-29b decreased ECM protein production in human corneal endothelial cells. Thus, miR-29 replacement therapy might be a new treatment strategy for FECD aimed at reducing pathologic production of ECM proteins in Descemet membrane.
authors.affiliation: *Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, MD; †Department of Ophthalmology, University of Tokyo, Tokyo, Japan; ‡Department of Ophthalmology, University of Cologne, Cologne, Germany; §Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; and ¶Inouye Eye Hospital, Tokyo, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Toyono
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Usui
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Villarreal
authors.forename: Laura
authors.initials: L
authors.lastname: Kallay
authors.forename: Mario
authors.initials: M
authors.lastname: Matthaei
authors.forename: Lucas M M
authors.initials: LM
authors.lastname: Vianna
authors.forename: Angela Y
authors.initials: AY
authors.lastname: Zhu
authors.forename: Masahiko
authors.initials: M
authors.lastname: Kuroda
authors.forename: Shiro
authors.initials: S
authors.lastname: Amano
authors.forename: Albert S
authors.initials: AS
authors.lastname: Jun
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D024141:Collagen Type IV / Q000235:genetics / Q000378:metabolism; D004728:Endothelium, Corneal / Q000378:metabolism* / Q000473:pathology; D005109:Extracellular Matrix / Q000235:genetics* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005642:Fuchs' Endothelial Dystrophy / Q000235:genetics* / Q000473:pathology; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007797:Laminin / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: C547475:COL4A1 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D024141:Collagen Type IV; D016326:Extracellular Matrix Proteins; D007797:Laminin; C532045:MIRN29a microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; C089141:laminin gamma 1