Methotrexate Inhibits NF‐κB Activity Via Long Intergenic (Noncoding) RNA–p21 Induction. Issue 11 (November 2014)

• Record Type: Journal Article
• Authors: Spurlock, Charles F.; Tossberg, John T.; Matlock, Brittany K.; Olsen, Nancy J.; Aune, Thomas M.
• Is Part Of: Arthritis & rheumatology. Volume 66:Issue 11(2014)
• Page Start: 2947
• Page End: 2957
• Publication Date: 2014-11
• Subjects: Arthritis -- Periodicals; Rheumatism -- Periodicals; 616.72
• DOI: 10.1002/art.38805
• Languages: English
• ISSNs: 2326-5191

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="art38805-sec-0001" sec-type="section"> <title>Objective</title> <p>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.</p> </sec> <sec id="art38805-sec-0002" sec-type="section"> <title>Methods</title> <p>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.</p> </sec> <sec id="art38805-sec-0003" sec-type="section"> <title>Results</title> <p>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 <italic>PRKDC</italic> 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 <italic>TP53</italic> and lincRNA‐p21 increased NF‐κB activity in cell lines. Decreased levels of lincRNA‐p21 did not alter <italic>NFKB1</italic> or <italic>RELA</italic> transcripts; rather, lincRNA‐p21 physically bound to <italic>RELA</italic> mRNA.</p> </sec> <sec id="art38805-sec-0004" sec-type="section"> <title>Conclusion</title> <p>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.</p> </sec> </abstract>