Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients

Abstract

Introduction: MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.

Methods: Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR.

Results: Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-alpha production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-alpha production.

Conclusions: Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-alpha production in patients with rheumatoid arthritis.

Figures

Figure 1

TNF-α treatment results in increased number of GWB in THP-1 and human PBMCs. (a) THP-1 cells were treated with 10 ng/ml TNF-α, IFN-α, IFN-β, IFN-γ, IL-12p70, M-CSF, IL-4, IL-10, or 25 ng/ml MCP-1 for 4 hours. IIF was performed using a human anti-GWB serum to detect GWB, and the number of GWB were counted using CellProfiler image analysis software. Average number of GWB per cell and SEM is shown. *P < 0.0001, as determined by one-way analysis of variance. (b) Human PBMCs were obtained from a healthy donor and isolated using Ficoll density-gradient centrifugation. The cells were then cultured for 4 hours in the presence of 1 ng/ml TNF-α. GWB were detected by IIF using rabbit anti-Rck/p54 antibodies. Average number of GWB and SEM is shown. *P < 0.0001, as determined by Mann-Whitney test. (c) IIF image of THP-1 and PBMCs treated with 10 ng/ml or 1 ng/ml TNF-α for 4 hours, respectively. GWB are shown in green, and nuclei are counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). Bar = 10 μm. GWP, GW or P bodies; IL, interleukin; IFN, interferon; IIF, indirect immunofluorescence; MCP, macrophage chemoattractant protein; M-CSF, macrophage colony-stimulating factor; PBMC, peripheral blood mononuclear cell; SEM, standard error of the mean; TNF, tumor necrosis factor.

Figure 2

RA patients exhibit aberrant expression of miR-146a, miR-155, miR-132 and miR-16 versus healthy controls. (a) RNA was isolated from healthy control individuals (n = 9), disease control individuals (n = 4), and RA patient (n = 17). PBMCs and relative expression levels of miR-146a, miR-155, miR-132, miR-16, and miRNA let-7a were analyzed by qRT-PCR using U44 RNA as an internal control. Average is indicated by bars. *P < 0.05, **P < 0.01, as determined by one-way analysis of variance. For RA patients, closed circles indicate patients undergoing anti-TNF-α therapy at time of sample collection, squares indicate MTX treatment, and open circles indicate other or no treatment. (b) Disease activity was determined for patients using CRP and ESR values and correlated with miRNA expression. Normal CRP and ESR values were classified as inactive disease (n = 3; patients 9, 12, and 14 in Table 1), and higher than normal CRP or ESR values were classified as active disease (n = 8; patients 1a, 1b, 2, 4, 5, 6, 10, and 15 in Table 1). Those patients with no or incomplete data for CRP/ESR values were omitted. *P < 0.05, as determined by t-test. (c) PBMCs were collected from patient RA-1 before (November 2007) and after (January 2008) MTX treatment and miRNA expression was examined using qRT-PCR. miRNA expression is largely consistent over time, with the exception of increased miR-16 expression. CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; miRNA, microRNA; MTX, methotrexate; PBMC, peripheral blood mononuclear cell; qRT-PCR, quantitative real-time RT-PCR; RA, rheumatoid arthritis; RT-PCR, reverse transcription polymerase chain reaction; TNF, tumor necrosis factor.

Figure 3

Monocyte/macrophage fraction of PBMCs exhibit increased miRNA expression compared with lymphocyte fraction. PBMCs were collected from RA patients and separated into monocyte/macrophage and lymphocyte populations by allowing the monocytes/macrophages to adhere to a tissue culture dish. (a) miRNA expression was examined using qRT-PCR. SEM is shown (n = 2 patients). (b) Average expression levels of miR-146a, miR-155, miR-132, and miR-16 are shown for monocyte and lymphocyte populations for two RA patients. *P < 0.02, as determined by Mann-Whitney test. SEM is shown. PBMC, peripheral blood mononuclear cell; qRT-PCR, quantitative real-time RT-PCR; RA, rheumatoid arthritis; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean.

Figure 4

TRAF6 and IRAK-1 expression levels are similar between RA patients, healthy controls, and disease controls. RNA was isolated from PBMCs from healthy control individuals (n = 9), disease control individuals (n = 4) and RA patients (n = 14), and mRNA expression levels of (a) TRAF6 and (b) IRAK-1 were analyzed using qRT-PCR. (c) PBMCs isolated from a healthy control individual and RA patient were incubated on glass slides for 1 hour at 37°C. The adhered cells were fixed and permeabilized in 3% paraformaldehyde and 0.5% Triton X-100, respectively. Protein levels of TRAF6 and IRAK-1 were analyzed by immunofluorescence using rabbit anti-TRAF6 and anti-IRAK-1 antibodies, and relative fluorescence was determined using Image J analysis software. SEM is shown; n > 20 cells. IRAK, IL-1 receptor-associated kinase; PBMC, peripheral blood mononuclear cell; qRT-PCR, quantitative real-time RT-PCR; RA, rheumatoid arthritis; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean; TRAF, tumor necrosis factor receptor-associated factor.

Figure 5

Knockdown of TRAF6 and/or IRAK-1 results in decreased TNF-α production in THP-1 cells. THP-1 cells were transfected with siRNA targeting TRAF6 and/or IRAK-1. (a) 48 hours after transfection, mRNA levels of TRAF6 and IRAK-1 were analyzed by qRT-PCR and normalized to mock transfected cells. SEM shown (n = 2). After knockdown of TRAF6 and/or IRAK-1 was confirmed by qRT-PCR, cells were treated with 1 μg/ml LPS for 24 hours and culture supernatants were collected. Multiplex assay was used to quantitatively detect (b) TNF-α and (c) MCP-1. IRAK, IL-1 receptor-associated kinase; LPS, lipopolysaccharide; MCP, macrophage chemoattractant protein; PBMC, peripheral blood mononuclear cell; qRT-PCR, quantitative real-time RT-PCR; RT-PCR, reverse transcription polymerase chain reaction; TNF, tumor necrosis factor; TRAF, tumor necrosis factor receptor-associated factor.