title: Methotrexate inhibits NF-κB activity via long intergenic (noncoding) RNA-p21 induction.
abstract: OBJECTIVE
  To determine interrelationships between the expression of long intergenic (noncoding) RNA-p21 (lincRNA-p21), NF-κB activity, and responses to methotrexate (MTX) in rheumatoid arthritis (RA) by analyzing patient blood samples and cell culture models.
  
  
  METHODS
  Expression levels of long noncoding RNA and messenger RNA (mRNA) were determined by quantitative reverse transcription-polymerase chain reaction. Western blotting and flow cytometry were used to quantify levels of intracellular proteins. Intracellular NF-κB activity was determined using an NF-κB luciferase reporter plasmid.
  
  
  RESULTS
  Patients with RA expressed reduced basal levels of lincRNA-p21 and increased basal levels of phosphorylated p65 (RelA), a marker of NF-κB activation. Patients with RA who were not treated with MTX expressed lower levels of lincRNA-p21 and higher levels of phosphorylated p65 compared with RA patients treated with low-dose MTX. In cell culture using primary cells and transformed cell lines, MTX induced lincRNA-p21 through a DNA-dependent protein kinase catalytic subunit (DNA PKcs)-dependent mechanism. Deficiencies in the levels of PRKDC mRNA in patients with RA were also corrected by MTX in vivo. Furthermore, MTX reduced NF-κB activity in tumor necrosis factor α-treated cells through a DNA PKcs-dependent mechanism via induction of lincRNA-p21. Finally, we observed that depressed levels of TP53 and lincRNA-p21 increased NF-κB activity in cell lines. Decreased levels of lincRNA-p21 did not alter NFKB1 or RELA transcripts; rather, lincRNA-p21 physically bound to RELA mRNA.
  
  
  CONCLUSIONS
  Our findings support a model whereby depressed levels of lincRNA-p21 in RA contribute to increased NF-κB activity. MTX decreases basal levels of NF-κB activity by increasing lincRNA-p21 levels through a DNA PKcs-dependent mechanism.
authors: Vanderbilt University School of Medicine, Nashville, Tennessee.
authors: Charles F
authors: CF
authors: Spurlock
authors: John T
authors: JT
authors: Tossberg
authors: Brittany K
authors: BK
authors: Matlock
authors: Nancy J
authors: NJ
authors: Olsen
authors: Thomas M
authors: TM
authors: Aune
mesh_terms: D000328:Adult; D018501:Antirheumatic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001172:Arthritis, Rheumatoid / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D008297:Male; D008727:Methotrexate / Q000494:pharmacology* / Q000627:therapeutic use; D008875:Middle Aged; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000378:metabolism*; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D019892:eIF-2 Kinase / Q000378:metabolism
chemical_list: D018501:Antirheumatic Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016328:NF-kappa B; D009687:Nuclear Proteins; D062085:RNA, Long Noncoding; D016159:Tumor Suppressor Protein p53; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D019892:eIF-2 Kinase; D008727:Methotrexate