Exploring the Extracellular Vesicle MicroRNA Expression Repertoire in Patients with Rheumatoid Arthritis and Ankylosing Spondylitis Treated with TNF Inhibitors

Abstract

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) belong to the most common inflammatory rheumatic diseases. MicroRNAs (miRNAs) are small 18-22 RNA molecules that function as posttranscriptional regulators. They are abundantly present within extracellular vesicles (EVs), small intercellular communication vesicles that can be found in bodily fluids and that have key functions in pathological and physiological pathways. Recently, EVs have gained much interest because of their diagnostic and therapeutic potential. Using NanoString profiling technology, the miRNA repertoire of serum EVs was determined and compared in RA and AS patients before and after anti-TNF therapy to assess its potential use as a diagnostic and prognostic biomarker. Furthermore, possible functional effects of those miRNAs that were characterized by the most significant expression changes were evaluated using in silico prediction algorithms. The analysis revealed a unique profile of differentially expressed miRNAs in RA and AS patient serum EVs. We identified 12 miRNAs whose expression profiles enabled differentiation between RA and AS patients before induction of anti-TNF treatment, as well as 4 and 14 miRNAs whose repertoires were significantly changed during the treatment in RA and AS patients, respectively. In conclusion, our findings suggest that extracellular vesicle miRNAs could be used as potential biomarkers associated with RA and AS response to biological treatment.

Figure 1

Serum EV microRNA expression in RA and AS patients before vs. after anti-TNF treatment. (a, d) Unsupervised hierarchical clustering analysis. Samples before (1, 3, and 5) vs. after (2, 4, and 6) anti-TNF therapy. (b, e) Volcano plots showing the relationship between fold change and significance for RA and AS patients before and after anti-TNF therapy. The horizontal dashed line indicates cutoff for significance p < 0.05 (−log10 p value > 1.3) and the vertical lines for fold change ≥ 1.5/≤−1.5. Significantly different miRNAs are highlighted in blue. (c) Heatmaps showing unsupervised hierarchical clustering of differentially expressed miRNAs in serum EVs of patients before (n = 3) vs. after Etanercept treatment. The colour scale indicates relative fold change (red: high; blue: low).

Figure 2

Serum EV microRNA expression in RA vs. AS patients before and three months after anti-TNF treatment. (a, d) Unsupervised hierarchical clustering analysis. RA vs. AS patients before biological treatment initialization and after agent administration. (b, e) Volcano plots showing the relationship between fold change and significance for RA vs. AS patients before and after anti-TNF therapy. The horizontal dashed line indicates cutoff for significance p < 0.05 (−log10 p value > 1.3) and the vertical lines for fold change ≥ 1.5/≤−1.5. Significantly different miRNAs are highlighted in blue. (c) Heatmaps showing unsupervised hierarchical clustering of differentially expressed miRNAs in serum EVs of RA vs. AS patients before (n = 3) and after treatment. The colour scale indicates relative fold change (red: high; blue: low).