title: MicroRNA-144 dysregulates the transforming growth factor-β signaling cascade and contributes to the development of bronchiolitis obliterans syndrome after human lung transplantation.
abstract: BACKGROUND
  Bronchiolitis obliterans syndrome (BOS), chronic lung allograft rejection, remains an impediment for the function of the transplanted organ. In this study, we defined the role of the microRNA (miRNA) miR-144 in fibroproliferation leading to BOS.
  
  
  METHODS
  Biopsy specimens were obtained from 20 lung transplant recipients with BOS((+)) and 19 without BOS((-)). Expression of miR-144 and its target, transforming growth factor-β (TGF-β)-induced factor homeobox 1(TGIF1), were analyzed by real-time polymerase chain reaction and Western blot. Overexpression of miR-144 and luciferase reporter genes were performed to elucidate miRNA-target interactions. The function of miR-144 was evaluated by transfecting fibroblasts and determining the response to TGF-β by analyzing Sma- and Mad-related family (Smads), fibroblast growth factor, TGF-β, and vascular endothelial growth factor. Smooth muscle actin-α-positive stress fibers and F-actin filaments in lung fibroblasts were analyzed by immunofluorescence.
  
  
  RESULTS
  Analysis of miR-144 in the biopsy specimens demonstrated 4.1 ± 0.8-fold increases in BOS(+) compared with BOS(-) patients, with a significant reduction in TGIF1 (3.6 ± 1.2-fold), a corepressor of Smads. In vitro transfection confirmed that over-expression of miR-144 results in a reduction in TGIF1 and an increase in SMAD2, SMAD4, fibroblast growth factor-6, TGF-β, and vascular endothelial growth factor. Increasing miR-144 by transfecting, increased smooth muscle actin-α and fibronectin, and knockdown of miR-144 diminished fibrogenesis in MRC-5 fibroblasts.
  
  
  CONCLUSIONS
  miR-144 is a critical regulator of the TGF-β signaling cascade and is over-expressed in lungs with BOS. Therefore, miR-144 is a potential target toward preventing fibrosis leading to BOS after lung transplant.
authors.affiliation: Departments of Surgery.
authors.forename: Zhongping
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Departments of Surgery.
authors.forename: Sabarinathan
authors.initials: S
authors.lastname: Ramachandran
authors.affiliation: Departments of Surgery.
authors.forename: Muthukumar
authors.initials: M
authors.lastname: Gunasekaran
authors.affiliation: Departments of Surgery.
authors.forename: Fangyu
authors.initials: F
authors.lastname: Zhou
authors.affiliation: Medicine.
authors.forename: Elbert
authors.initials: E
authors.lastname: Trulock
authors.affiliation: Departments of Surgery.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kreisel
authors.affiliation: Medicine.
authors.forename: Ramsey
authors.initials: R
authors.lastname: Hachem
authors.affiliation: Departments of Surgery; Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri. Electronic address: kumart@wustl.edu.
authors.forename: Thalachallour
authors.initials: T
authors.lastname: Mohanakumar
mesh_terms: D015153:Blotting, Western; D001989:Bronchiolitis Obliterans / Q000517:prevention & control*; D005260:Female; D005346:Fibroblast Growth Factors / Q000032:analysis; D005347:Fibroblasts / Q000737:chemistry; D005355:Fibrosis / Q000517:prevention & control; D005455:Fluorescent Antibody Technique; D018398:Homeodomain Proteins / Q000032:analysis; D006801:Humans; D008168:Lung / Q000737:chemistry; D016040:Lung Transplantation; D008297:Male; D035683:MicroRNAs / Q000502:physiology*; D008875:Middle Aged; D009132:Muscles / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000032:analysis; D051785:Smad Proteins / Q000032:analysis; D051899:Smad2 Protein / Q000032:analysis; D051901:Smad4 Protein / Q000032:analysis; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis
chemical_list: D018398:Homeodomain Proteins; C554293:MIRN144 microRNA, human; D035683:MicroRNAs; D012097:Repressor Proteins; D051785:Smad Proteins; D051899:Smad2 Protein; D051901:Smad4 Protein; C097504:TGIF1 protein, human; D016212:Transforming Growth Factor beta; D042461:Vascular Endothelial Growth Factor A; D005346:Fibroblast Growth Factors
keywords: TGF-β.; bronchiolitis obliterans syndrome; lung transplantation; microRNA