Methotrexate inhibits NF-κB activity via long intergenic (noncoding) RNA-p21 induction

Area of Science:

• Molecular Biology
• Immunology
• Rheumatology

Background:

• Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation.
• Nuclear factor-kappa B (NF-κB) signaling plays a critical role in RA pathogenesis.
• Long intergenic noncoding RNA-p21 (lincRNA-p21) has emerged as a potential regulator in inflammatory conditions.

Purpose of the Study:

• To investigate the relationship between lincRNA-p21 expression, NF-κB activity, and response to methotrexate (MTX) in rheumatoid arthritis (RA).
• To elucidate the molecular mechanisms underlying these interactions in both patient samples and cell models.

Main Methods:

• Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for gene expression analysis.
• Western blotting and flow cytometry for protein level quantification.
• NF-κB luciferase reporter assays to measure NF-κB activity.
• Analysis of patient blood samples and cell culture models.

Main Results:

• Patients with RA exhibited lower basal lincRNA-p21 and higher phosphorylated p65 (NF-κB marker) levels.
• Methotrexate (MTX) treatment in RA patients correlated with increased lincRNA-p21 and reduced NF-κB activity.
• MTX induced lincRNA-p21 via a DNA-dependent protein kinase catalytic subunit (DNA PKcs)-dependent pathway, which also reduced NF-κB activity.
• lincRNA-p21 directly bound to RELA mRNA, influencing NF-κB activity.

Conclusions:

• Depressed lincRNA-p21 levels in RA contribute to heightened NF-κB activation.
• MTX treatment ameliorates RA by upregulating lincRNA-p21 through a DNA PKcs-dependent mechanism, consequently suppressing NF-κB activity.