title: MiR-20a regulates ASK1 expression and TLR4-dependent cytokine release in rheumatoid fibroblast-like synoviocytes.
abstract: OBJECTIVE
  To evaluate whether miR-20a belonging to the cluster miR-17-92 is a negative regulator of inflammation in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) by modulating expression of apoptosis signal-regulating kinase (ASK) 1, a key component of the toll-like receptors 4 pathway, upstream of p38 mitogen-activated protein kinase.
  
  
  METHODS
  Evaluation of miR-20a and ASK1 mRNA was performed by RT-qPCR. ASK1 protein expression was assessed by western blotting. Overexpression of miR-20a was performed by transfection of RA FLS and THP-1 cells with miR-20a mimics. Interleukin (IL)-6, CXCL-10, IL-1β and TNF-α release were measured by ELISA. The role of miR-20a in vivo was assessed by IL-6 release from macrophages obtained from mice injected intraperitoneally with vectorised miR-20a mimics.
  
  
  RESULTS
  We showed that stimulation of RA FLS with lipopolysacharide (LPS) and bacterial lipoproteins (BLP) induces a drop in expression of miR-20a and that this decrease is associated with an upregulation of ASK1 expression. Using transfection of Ask1 3'UTR reporters, we demonstrate that Ask1 is a direct target of miR-20a. Overexpression of miR-20a led to a global decrease in ASK1 protein in BLP- and LPS-activated cells indicating that miR-20a regulates the expression of ASK1 at the translational level. Transfection of miR-20a mimics decreases IL-6 and CXCL10 release by RA FLS and IL-1β and TNF-α by activated THP-1 cells but only in response to LPS. Last, injection of vectorised miR-20a mimics to mice led to a global decrease in ASK1 protein expression and IL-6 secretion in LPS-activated macrophages.
  
  
  CONCLUSIONS
  Our data point toward an important role for miR-20a in the regulation of pro-inflammatory cytokines release, by controlling ASK1 expression in RA FLS.
authors.affiliation: ImmunoRhumatologie Moléculaire, INSERM UMR_S 1109, Université de Strasbourg, Strasbourg, Cedex, France.
authors.forename: Lucas
authors.initials: L
authors.lastname: Philippe
authors.forename: Ghada
authors.initials: G
authors.lastname: Alsaleh
authors.forename: Angélique
authors.initials: A
authors.lastname: Pichot
authors.forename: Eleonore
authors.initials: E
authors.lastname: Ostermann
authors.forename: Guy
authors.initials: G
authors.lastname: Zuber
authors.forename: Benoit
authors.initials: B
authors.lastname: Frisch
authors.forename: Jean
authors.initials: J
authors.lastname: Sibilia
authors.forename: Sébastien
authors.initials: S
authors.lastname: Pfeffer
authors.forename: Seiamak
authors.initials: S
authors.lastname: Bahram
authors.forename: Dominique
authors.initials: D
authors.lastname: Wachsmann
authors.forename: Philippe
authors.initials: P
authors.lastname: Georgel
mesh_terms: D000818:Animals; D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000503:physiopathology*; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis*; D013583:Synovial Membrane / Q000166:cytology* / Q000378:metabolism / Q000503:physiopathology
chemical_list: D016207:Cytokines; C534451:MIRN20a microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human; C482859:Map3k5 protein, mouse
keywords: Cytokines; Fibroblasts; Inflammation; Rheumatoid Arthritis