Background Recent epigenetic studies reveal the pathogenic role of micro-ribonucleic acids (microRNAs) and their targets in the inflammatory process in rheumatoid arthritis (RA). miRNAs play crucial role in controlling and modulating immunity and their abnormal expression has been linked to the deregulated function of regulatory T cells, to the chronic synovial inflammation and bone destruction^1–3.

Objectives To perform a multimarker analysis of synovial fluid (SF) expression levels of miR-146a, miR-155 and miR-223 in RA patients in regard to their role as diagnostic biomarkers.

Methods Total RNA was isolated from the SF of 48 RA patients and 11 healthy controls (HCs) and expression levels of miR-146a, miR-155 and miR-223 were determined by quantitative real-time polymerase chain reaction (qPCR), SybrGreen technology. Relative changes of gene expression levels of the miRNAs were calculated by 2^-ΔΔCtmethod and SPSS were used for statistical analysis. RNU6B gene was used as a reference control for normalization. Receiver operating characteristic (ROC) curve analysis using RQ values was constructed in order to evaluate the diagnostic accuracy of these miRNAs in SF for distinguishing RA patients from HCs.

Results miR-146a, miR-155 and miR-223 showed overexpression in RA SF compared to HCs (in 70.83%, 79.17% and 79.17% of the patients, respectively) and could be used to differentiate RA patients from HCs (p=4.8x10^-4, p=8x10^-5 and p=2.8x10^-4, respectively). The ROC curve analysis showed diagnostic accuracy for miR-146a with area under the curve (AUC)=0.769, 95% CI=0.600÷0.938, p=0.006, with sensitivity of 75% and specificity of 72.3%; AUC for miR-155 was 0.858 (95% CI=0.757÷0.959, p=2.3x10^-4) with sensitivity of 81.3% and specificity of 81.8%; AUC for miR-223=0.841 (95% CI=0.724÷0.958, p=4.6x10^-4), with sensitivity of 87.5% and specificity of 72.7%. The diagnostic accuracy improved when performing a multimarker analysis with AUC for the combination of miR-146a and miR-155=0.871 (0.776–0.967, p=1.4x10^-4), AUC for miR-146a and miR-223=0.867 (0.753–0.982, p=1.6x10^-4), AUC for miR-155 and miR-223=0.907 (0.823–0.991, p=2.6x10^-5). AUC for the combination of all three miRNAs was 0.915 (0.840–0.990, p=2.02x10^-5) with 89.6% sensitivity and 81.8% specificity.

Conclusions Local miRNA expression levels could serve as diagnostic biomarkers in RA patients. The multimarker analysis of the expression levels of miR-146a, miR-155 and miR-223 in SF has better diagnostic accuracy than their single use in the clinical practice.

References

1. Chen XM, Huang QC, Yang SL, Chu YL, Yan YH, Han L et al. Role of Micro RNAs in the Pathogenesis of Rheumatoid Arthritis. Medicine (Baltimore). 2015; 94(31):e1326.

2. Churov AV, Oleinik EK, Knip M. MicroRNAs in rheumatoid arthritis: altered expression and diagnostic 2. potential. Autoimmun Rev. 2015; 14(11):1029–37.

3. Murata K, Yoshitomi H, Tanida S, Ishikawa M, Nishitani K, Ito H, Nakamura T. Plasma and synovial fluid microRNAs as potential biomarkers of rheumatoid arthritis and osteoarthritis. Arthritis Res Ther. 2010;12(3):R86.

References

Acknowledgements The study was supported by Grant 14-D/2012 and 55/2013 funded by MU-Sofia.

Disclosure of Interest None declared