Circulating miRNAs as potential biomarkers of therapy effectiveness in rheumatoid arthritis patients treated with anti-TNFα

Introduction: The advent of anti-tumor necrosis factor alpha (anti-TNF alpha) drugs has considerably improved medical management in rheumatoid arthritis (RA) patients, although it has been reported to be ineffective in a fraction of them. MicroRNAs (miRNAs) are small, non-coding RNAs that act as fine-tuning regulators of gene expression. Targeting miRNAs by gain or loss of function approaches have brought therapeutic effects in various disease models. The aim of this study was to investigate serum miRNA levels as predictive biomarkers of response to anti-TNF alpha therapy in RA patients. Methods: In total, 95 RA patients undergoing anti-TNF alpha/disease-modifying antirheumatic drugs (anti-TNF alpha/DMARDs) combined treatments were enrolled. Serum samples were obtained at 0 and 6 months and therapeutic efficacy was assessed. miRNAs were isolated from the serum of 10 patients before and after anti-TNF alpha/DMARDs combination therapy, cDNA transcribed and pooled, and human serum miRNA polymerase chain reaction (PCR) arrays were performed. Subsequently, selected miRNAs were analyzed in a validation cohort consisting of 85 RA patients. Correlation studies with clinical and serological variables were also performed. Results: Ninety percent of RA patients responded to anti-TNF alpha/DMARDs combination therapy according to European League Against Rheumatism (EULAR) criteria. Array analysis showed that 91% of miRNAS were overexpressed and 9% downregulated after therapy. Functional classification revealed a preponderance of target mRNAs involved in reduction of cells maturation - especially on chondrocytes - as well as in immune and inflammatory response, cardiovascular disease, connective tissue and musculoskeletal system. Six out of ten miRNAs selected for validation were found significantly upregulated by anti-TNF alpha/DMARDs combination therapy (miR-16-5p, miR-23-3p, miR125b-5p, miR-126-3p, miRN-146a-5p, miR-223-3p). Only responder patients showed an increase in those miRNAs after therapy, and paralleled the reduction of TNF alpha, interleukin (IL)-6, IL-17, rheumatoid factor (RF), and C-reactive protein (CRP). Correlation studies demonstrated associations between validated miRNAs and clinical and inflammatory parameters. Further, we identified a specific plasma miRNA signature (miR-23 and miR-223) that may serve both as predictor and biomarker of response to anti-TNF alpha/DMARDs combination therapy. Conclusions: miRNA levels in the serum of RA patients before and after anti-TNF alpha/DMARDs combination therapy are potential novel biomarkers for predicting and monitoring therapy outcome.