title: Inhibition and role of let-7d in idiopathic pulmonary fibrosis.
abstract: BACKGROUND
  Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation.
  
  
  OBJECTIVE
  To determine changes in expression and role of microRNAs in IPF.
  
  
  METHODS
  RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry.
  
  
  RESULTS
  Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells.
  
  
  CONCLUSIONS
  Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).
authors.affiliation: University of Pittsburgh Medical Center, NW 628 MUH, 3459 5th Avenue, Pittsburgh, PA 15261, USA. kaminskin@upmc.edu
authors.forename: Kusum V
authors.initials: KV
authors.lastname: Pandit
authors.forename: David
authors.initials: D
authors.lastname: Corcoran
authors.forename: Hanadie
authors.initials: H
authors.lastname: Yousef
authors.forename: Manohar
authors.initials: M
authors.lastname: Yarlagadda
authors.forename: Argyris
authors.initials: A
authors.lastname: Tzouvelekis
authors.forename: Kevin F
authors.initials: KF
authors.lastname: Gibson
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Konishi
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Yousem
authors.forename: Mandal
authors.initials: M
authors.lastname: Singh
authors.forename: Daniel
authors.initials: D
authors.lastname: Handley
authors.forename: Thomas
authors.initials: T
authors.lastname: Richards
authors.forename: Moises
authors.initials: M
authors.lastname: Selman
authors.forename: Simon C
authors.initials: SC
authors.lastname: Watkins
authors.forename: Annie
authors.initials: A
authors.lastname: Pardo
authors.forename: Ahmi
authors.initials: A
authors.lastname: Ben-Yehudah
authors.forename: Demosthenes
authors.initials: D
authors.lastname: Bouros
authors.forename: Oliver
authors.initials: O
authors.lastname: Eickelberg
authors.forename: Prabir
authors.initials: P
authors.lastname: Ray
authors.forename: Panayiotis V
authors.initials: PV
authors.lastname: Benos
authors.forename: Naftali
authors.initials: N
authors.lastname: Kaminski
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation; D004847:Epithelial Cells / Q000378:metabolism; D025743:HMGA2 Protein / Q000378:metabolism; D006801:Humans; D054990:Idiopathic Pulmonary Fibrosis / Q000378:metabolism* / Q000473:pathology; D017403:In Situ Hybridization; D008168:Lung / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011650:Pulmonary Alveoli / Q000378:metabolism; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000502:physiology; D014746:Vimentin / Q000378:metabolism
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D015820:Cadherins; D025743:HMGA2 Protein; D035683:MicroRNAs; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C470944:S100a4 protein, mouse; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D014746:Vimentin; C487931:mirnlet7 microRNA, human
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