title: The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13.
abstract: OBJECTIVE
  To identify differentially expressed microRNAs (miRNAs) in human osteoarthritic (OA) cartilage and bone tissue and to determine their relevance to chondrocyte function.
  
  
  METHODS
  Cartilage and bone was obtained from OA patients who underwent total knee joint replacement surgery or from post-mortem patients with no previous history of OA. MiRNA expression was quantified by real-time PCR (RT-PCR). Functional pathway analysis of miRNA was performed using Ingenuity Pathway Analysis. Primary chondrocytes were isolated by collagenase digestion and transfected with miRNA mimics and miRNA inhibitors using cationic lipid. Tumour Necrosis Factor-alpha (TNF-alpha) and Matrix metalloprotease 13 (MMP13) protein levels were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).
  
  
  RESULTS
  In total we identified 17 miRNA that showed greater than 4-fold differential expression between OA and normal cartilage, and 30 miRNA that showed greater than 4-fold differential expression in OA bone. Functional pathway analysis of the predicted gene targets for miR-9, miR-98, which were upregulated in both OA bone and cartilage tissue, and miR-146, which was downregulated in OA cartilage, suggested that these miRNA mediate inflammatory functions and pathways. Over-expression of miR-9, miR-98 or miR-146 in isolated human chondrocytes reduced interleukin-1 beta (IL-1 beta) induced TNF-alpha production. Furthermore, inhibition and over-expression of miR-9 modulated MMP13 secretion.
  
  
  CONCLUSIONS
  We have identified a number of differentially expressed miRNAs in late-stage human OA cartilage and bone. Functional analysis of miR-9, miR-98 and miR-146 in primary chondrocytes suggests a role in mediating the IL-1 beta induced production of TNF-alpha. MiR-9, upregulated in OA tissue, was found to inhibit secretion of the collagen type II-targeting metalloproteinase MMP13 in isolated human chondrocytes.
authors.affiliation: Disease Science, Respiratory and Inflammation Research Area, AstraZeneca Alderley Park, Macclesfield SK10 4TG, United Kingdom. simon.w.jones@astrazeneca.com
authors.forename: S W
authors.initials: SW
authors.lastname: Jones
authors.forename: G
authors.initials: G
authors.lastname: Watkins
authors.forename: N
authors.initials: N
authors.lastname: Le Good
authors.forename: S
authors.initials: S
authors.lastname: Roberts
authors.forename: C L
authors.initials: CL
authors.lastname: Murphy
authors.forename: S M V
authors.initials: SM
authors.lastname: Brockbank
authors.forename: M R C
authors.initials: MR
authors.lastname: Needham
authors.forename: S J
authors.initials: SJ
authors.lastname: Read
authors.forename: P
authors.initials: P
authors.lastname: Newham
mesh_terms: D000368:Aged; D002358:Cartilage, Articular / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D006801:Humans; D018836:Inflammation Mediators / Q000502:physiology; D053509:Matrix Metalloproteinase 13 / Q000096:biosynthesis*; D035683:MicroRNAs / Q000235:genetics / Q000502:physiology*; D008875:Middle Aged; D020370:Osteoarthritis, Knee / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D018836:Inflammation Mediators; D035683:MicroRNAs; D014409:Tumor Necrosis Factor-alpha; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13