title: Down-regulation of microRNA-34a* in rheumatoid arthritis synovial fibroblasts promotes apoptosis resistance.
abstract: OBJECTIVE
  To investigate the expression and effect of the microRNA-34 (miR-34) family on apoptosis in rheumatoid arthritis synovial fibroblasts (RASFs).
  
  
  METHODS
  Expression of the miR-34 family in synovial fibroblasts with or without stimulation with Toll-like receptor (TLR) ligands, tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), hypoxia, or 5-azacytidine was analyzed by real-time polymerase chain reaction (PCR). Promoter methylation was studied by combined bisulfite restriction analysis. The effects of overexpression and silencing of miR-34a and miR-34a* on apoptosis were analyzed by annexin V/propidium iodide staining. Production of X-linked inhibitor of apoptosis protein (XIAP) was assessed by real-time PCR and immunohistochemistry analysis. Reporter gene assay was used to study the signaling pathways of miR-34a*.
  
  
  RESULTS
  Basal expression levels of miR-34a* were found to be reduced in synovial fibroblasts from RA patients compared to osteoarthritis patients, whereas levels of miR-34a, miR-34b/b*, and miR-34c/c* did not differ. Neither TNFα, IL-1β, TLR ligands, nor hypoxia altered miR-34a* expression. However, we demonstrated that the promoter of miR-34a/34a* was methylated and showed that transcription of the miR-34a duplex was induced upon treatment with demethylating agents. Enforced expression of miR-34a* led to an increased rate of FasL- and TRAIL-mediated apoptosis in RASFs. Moreover, levels of miR-34a* were highly correlated with expression of XIAP, which was found to be up-regulated in RA synovial cells. Finally, we identified XIAP as a direct target of miR-34a*.
  
  
  CONCLUSIONS
  Our data provide evidence of a methylation-specific down-regulation of proapoptotic miR-34a* in RASFs. Decreased expression of miR- 34a* results in up-regulation of its direct target XIAP, thereby contributing to resistance of RASFs to apoptosis.
authors.affiliation: University Hospital Zurich, Zurich, Switzerland.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Niederer
authors.forename: Michelle
authors.initials: M
authors.lastname: Trenkmann
authors.forename: Caroline
authors.initials: C
authors.lastname: Ospelt
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Karouzakis
authors.forename: Michel
authors.initials: M
authors.lastname: Neidhart
authors.forename: Joanna
authors.initials: J
authors.lastname: Stanczyk
authors.forename: Christoph
authors.initials: C
authors.lastname: Kolling
authors.forename: Renate E
authors.initials: RE
authors.lastname: Gay
authors.forename: Michael
authors.initials: M
authors.lastname: Detmar
authors.forename: Steffen
authors.initials: S
authors.lastname: Gay
authors.forename: Astrid
authors.initials: A
authors.lastname: Jüngel
authors.forename: Diego
authors.initials: D
authors.lastname: Kyburz
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D001374:Azacitidine / Q000494:pharmacology; D002478:Cells, Cultured; D015536:Down-Regulation; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D000860:Hypoxia / Q000378:metabolism / Q000473:pathology; D053583:Interleukin-1beta / Q000494:pharmacology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D013583:Synovial Membrane / Q000378:metabolism* / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D053583:Interleukin-1beta; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D014409:Tumor Necrosis Factor-alpha; D051636:X-Linked Inhibitor of Apoptosis Protein; D001374:Azacitidine