Comprehensive microRNA Analysis Identifies miR-24 and miR-125a-5p as Plasma Biomarkers for Rheumatoid Arthritis

<div><p>MicroRNAs (miRNAs) are present in human plasma and known as a non-invasive biomarker for cancer detection. Our study was designed to identify plasma miRNAs specific for rheumatoid arthritis (RA) by a comprehensive array approach. We performed a systematic, array-based miRNA analysis on plasma samples from three RA patients and three healthy controls (HCs). Plasma miRNAs with more than four times change or with significant (<i>P</i><0.05) change in expression, or detectable only in RA plasma, were confirmed with plasma from eight RA patients and eight HCs using real-time quantitative PCR. Consistently detectable miRNAs that were significantly different between RA patients and HCs were chosen for further validation with 102 RA patients and 104 HCs. The area under curves (AUC) were calculated after plotting the receiver operating characteristic (ROC) curves. To determine if these miRNAs are specific for RA, the concentrations of these miRNAs were analyzed in 24 patients with osteoarthritis (OA), and 11 patients with systemic lupus erythematosus (SLE). The array analysis and the subsequent confirmation in larger patient cohort identified significant alterations in plasma levels of seven miRNAs. The highest AUC was found for miR-125a-5p, followed in order by miR-24 and miR-26a. Multivariable logistic regression analysis showed that miR-24, miR-30a-5p, and miR-125a-5p were crucial factors for making detection model of RA and provided a formula for <i>E</i>stimated <i>P</i>robability of <i>RA</i> by plasma <i>M</i>iRNA (ePRAM), employing miR-24, miR-30a-5p and miR-125a-5p, which showed increased diagnostic accuracy (AUC: 0.89). The level of miR-24, miR-125a-5p, and ePRAM in OA and SLE patients were lower than that in RA. There was no significant difference in detection for anti-citrullinated protein antibody (ACPA)-positive and ACPA-negative RA patients. These results suggest that the plasma concentrations of miR-24 and miR-125a-5p, and ePRAM are potential diagnostic markers of RA even if patients were ACPA-negative.</p></div

Suppressor of TCR signaling-2 (STS-2) suppresses arthritis development in mice

<p><b>Objectives:</b> Suppressor of TCR signaling-2 (STS-2) is one of the RA susceptibility genes identified in genome-wide association studies (GWAS). We tried to verify the involvement of STS-2 on the development of autoimmune arthritis in a mouse model.</p> <p><b>Methods:</b> STS-2 knock-out (KO) and wild type (WT) mice were immunized with chicken type II collagen (CII). For CD4⁺ helper T cell (Th) subset analysis, intracellular cytokines in splenocytes and lymph node cells were stained and analyzed by flow cytometry. Regulatory T cell (Treg) function was analyzed by co-culturing effector CD4⁺T cells and Tregs collected from non-immunized mice.</p> <p><b>Results:</b> CII-immunized STS-2 KO mice developed arthritis more frequently than WT mice. Although the T cell activation profile and Th subset in spleen and LNs were similar between STS-2 KO and WT mice, STS-2 KO mice showed increased IL-2-producing CD4⁺T cells in spleen when compared with WT mice. Accordingly, STS-2 KO CD4⁺T cells promoted IL-2 production by TCR stimulation. However, STS-2 KO Tregs normally suppressed T cell proliferation.</p> <p><b>Conclusion:</b> We proved that STS-2 is involved in the arthritis development by collagen-induced arthritis. Higher IL-2 production from STS-2 KO T cells is suggested to have a main pathogenic role in arthritis development.</p

A flowchart illustrates how we analyzed plasma miRNAs.

<p>We performed a systematic, array-based miRNA analysis on plasma samples from three rheumatoid arthritis (RA) patients and three healthy controls (HCs). Plasma miRNAs with more than four times change or with significant (P<0.05) change in expression, or detectable only in RA plasma, were confirmed with plasma from eight RA patients and eight HCs using real-time quantitative PCR (qRT-PCR). Eleven differently expressed miRNAs and one normalizer miRNA (miR-30a-5p) were chosen for further validation with 102 RA patients and 104 HCs. The concentrations of two specific miRNAs and one normalizer miRNA were measured in 24 patients with osteoarthritis (OA), and 11 patients with systemic lupus erythematosus (SLE). We also tried to find out miRNA that could be used for normalizer with geNorm and NormFinder using the results of microarray. miR-30a-5p was a candidate normalizer.</p

Evaluation of candidate miRNAs.

<p><b>A:</b> Clinical features of patients with OA and SLE who contributed plasma. <b>B:</b> Plasma concentrations of miR-24, miR-125a-5p, and ePRAM in patients with osteoarthritis (OA) and systemic lupus arthritis (SLE). The green areas indicate the ranges in which patients are diagnosed as RA positive. <b>C:</b> Histogram of plasma concentrations of each miRNA in anti-citrullinated protein antibody (ACPA)-negative RA patients, ACPA-positive RA patients, and HCs. The green panels indicate the cutoff value. <b>D:</b> Sensitivity of each miRNA test in ACPA-positive and ACPA-negative RA patients. Data are shown as mean ± standard deviation. SLEDAI = SLE Disease Activity Index.</p

Clinical details of the patients with rheumatoid arthritis (RA) and healthy controls (HCs).

<p>All values are reported as mean ± standard deviation.</p><p>ACPA = anti-citrullinated protein antibody; ESR = erythrocyte sedimentation ratio; MMP3 =  metalloproteinase-3, DAS28 = 28-joint Disease Activity Score, VAS = visual analogue scale of general health, DMARDs = Disease Modifying Anti-rheumatic Drugs, NA = not applicable.</p

Prognostic Significance of Anti-Aminoacyl-tRNA Synthetase Antibodies in Polymyositis/Dermatomyositis-Associated Interstitial Lung Disease: A Retrospective Case Control Study

<div><p>Background</p><p>In polymyositis/dermatomyositis (PM/DM), anti-aminoacyl-tRNA synthetase (ARS) antibodies are closely associated with interstitial lung disease (ILD), a frequent pulmonary complication. However, the clinical significance of anti-ARS antibodies is not well established.</p><p>Objective</p><p>We aimed to evaluate the clinical significance of anti-ARS antibodies in PM/DM-ILD patients.</p><p>Methods</p><p>Forty-eight consecutive PM/DM-ILD patients were studied retrospectively. Anti-ARS antibodies were screened by ELISA and confirmed by RNA immunoprecipitation test. Medical records, high-resolution computed tomography images, and surgical lung biopsy specimens were compared between ARS-positive (ARS group) and ARS-negative patients (non-ARS group).</p><p>Results</p><p>Anti-ARS antibodies were detected in 23 of 48 patients (48%). Radiologically, nonspecific interstitial pneumonia (NSIP) pattern was observed more frequently in the ARS group than in the non-ARS group (73.9% vs. 40%, <i>P</i> = 0.02). Pathologically, NSIP was the most frequent in both groups. Ten-year survival rate was also significantly higher in the ARS group than in the non-ARS group (91.6% vs. 58.7%, <i>P</i> = 0.02). Univariate Cox hazards analysis revealed that the presence of anti-ARS antibodies was associated with better prognosis (HR = 0.34, 95% CI 0.08–0.80; <i>P</i> = 0.01).</p><p>Conclusions</p><p>The presence of anti-ARS antibodies is a possible prognostic marker in patients with PM/DM-ILD.</p></div

Plasma miRNA profiling using miRNA array.

<p><b>A:</b> Microarray analysis for miRNA levels was performed with RNA isolated from the plasma of three patients with rheumatoid arthritis (RA) and three healthy controls (HCs). The differences of the -ΔCt averages of each miRNA between RA and HC are demonstrated in z axis. <b>B:</b> Background of the individuals who contributed the plasma samples. <b>C:</b> miRNAs with at least a four-fold differential expression, with the expression only in RA, and with significantly different levels between RA patients and HCs (<i>P</i><0.05). PSL = prednisolone, MTX = methotrexate, MZR = mizoribine, mPSL = methylprednisolone, ^¶ = miRNAs listed in other categories, N.A. = not assessed.</p

Treatment and outcome.

<p>Data are presented as n (%), median (range).</p><p>*<i>P</i> < 0.05</p><p>Treatment and outcome.</p

Plasma miR-24, miR-26a and miR-125a-5p differentiated RA from HC with high specificity.

<p><b>A:</b> Plasma concentrations of eight miRNAs for candidate biomarker and miR-30a-5p for candidate normalizer in RA patients (n = 102) and HC (n = 104). Data are shown as mean ± standard deviation. Area under curve (AUC) was calculated after plotting receiver operating characteristic (ROC) curve. <b>B:</b> ROC curve analyses of miR-24 (left panel), miR-26a (middle panel) and miR-125a-5p (right panel), which showed the highest values for AUC. <b>C:</b> Histograms of plasma concentrations of miR-24 (left panel), miR-26a (middle panel), and miR-125a-5p (right panel). The green panels indicate the cutoff value. <b>D:</b> Sensitivity and specificity of each miRNA test for RA. Cutoff value with higher specificity was selected between the maximum and the maximum minus 0.03 after the maximum of the sum of true positive rate and false positive rate were calculated.</p

Univariate Cox hazards analysis for survival.

<p>*<i>P</i> < 0.05</p><p>HR, hazard ratio; 95%CI, 95% confidence interval; ILD, interstitial lung disease; PM, polymyositis; DM, dermatomyositis; CADM, clinically amyopathic dermatomyositis; PaO₂, arterial oxygen pressure; %FVC, predicted forced vital capacity.</p><p>Univariate Cox hazards analysis for survival.</p