title: Impact of gastro-oesophageal reflux on microRNA expression, location and function.
abstract: BACKGROUND
  Ulceration of the oesophageal squamous mucosa (ulcerative oesophagitis) is a pathological manifestation of gastro-oesophageal reflux disease, and is a major risk factor for the development of Barrett's oesophagus. Barrett's oesophagus is characterised by replacement of reflux-damaged oesophageal squamous epithelium with a columnar intestinal-like epithelium. We previously reported discovery of microRNAs that are differentially expressed between oesophageal squamous mucosa and Barrett's oesophagus mucosa. Now, to better understand early steps in the initiation of Barrett's oesophagus, we assessed the expression, location and function of these microRNAs in oesophageal squamous mucosa from individuals with ulcerative oesophagitis.
  
  
  METHODS
  Quantitative real-time PCR was used to compare miR-21, 143, 145, 194, 203, 205 and 215 expression levels in oesophageal mucosa from individuals without pathological gastro-oesophageal reflux to individuals with ulcerative oesophagitis. Correlations between microRNA expression and messenger RNA differentiation markers BMP-4, CK8 and CK14 were analyzed. The cellular localisation of microRNAs within the oesophageal mucosa was determined using in-situ hybridisation. microRNA involvement in proliferation and apoptosis was assessed following transfection of a human squamous oesophageal mucosal cell line (Het-1A).
  
  
  RESULTS
  miR-143, miR-145 and miR-205 levels were significantly higher in gastro-oesophageal reflux compared with controls. Elevated miR-143 expression correlated with BMP-4 and CK8 expression, and elevated miR-205 expression correlated negatively with CK14 expression. Endogenous miR-143, miR-145 and miR-205 expression was localised to the basal layer of the oesophageal epithelium. Transfection of miR-143, 145 and 205 mimics into Het-1A cells resulted in increased apoptosis and decreased proliferation.
  
  
  CONCLUSIONS
  Elevated miR-143, miR-145 and miR-205 expression was observed in oesophageal squamous mucosa of individuals with ulcerative oesophagitis. These miRNAs localised to the basal layer of the oesophageal epithelium. They reduced proliferation and increased apoptosis, and may play roles in regulating epithelial restoration in response to injury caused by gastro-oesophageal reflux.
authors.affiliation: Department of Surgery, Flinders University, Flinders Medical Centre, Bedford Park, SA 5042, Australia.
authors.forename: Cameron M
authors.initials: CM
authors.lastname: Smith
authors.forename: Michael Z
authors.initials: MZ
authors.lastname: Michael
authors.forename: David I
authors.initials: DI
authors.lastname: Watson
authors.forename: Grace
authors.initials: G
authors.lastname: Tan
authors.forename: David St J
authors.initials: DS
authors.lastname: Astill
authors.forename: Richard
authors.initials: R
authors.lastname: Hummel
authors.forename: Damian J
authors.initials: DJ
authors.lastname: Hussey
mesh_terms: D017209:Apoptosis; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism; D016022:Case-Control Studies; D049109:Cell Proliferation; D002478:Cells, Cultured; D004947:Esophagus / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D005764:Gastroesophageal Reflux / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D006801:Humans; D053547:Keratin-14 / Q000378:metabolism; D053533:Keratin-8 / Q000378:metabolism; D035683:MicroRNAs / Q000502:physiology*; D008875:Middle Aged; D009092:Mucous Membrane / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology
chemical_list: D055415:Bone Morphogenetic Protein 4; D053547:Keratin-14; D053533:Keratin-8; D035683:MicroRNAs; C525809:BMP4 protein, human; C542359:MIRN143 microRNA, human; C541010:MIRN145 microRNA, human; C534425:MIR205, human