Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and joint destruction. Cell-derived small extracellular vesicles (sEV) mediate cell-to-cell communication in the synovial microenvironment by carrying microRNAs (miRs), a class of small non-coding RNAs. Herein, we report that sEV from synovial fluid promote osteoclast differentiation which is attributed to high levels of extracellular miR-574-5p. Moreover, we demonstrate for the first time that enhanced osteoclast maturation is mediated by Toll-like receptor (TLR) 7/8 signaling which is activated by miR-574-5p binding. This is a novel mechanism by which sEV and miRs contribute to RA pathogenesis and indicate that pharmacological inhibition of extracellular miR-574-5p might offer new therapeutic strategies to protect osteoclast-mediated bone destruction in RA.

Extracellular microRNAs (miRs) have been proposed as important blood‐based biomarkers for several diseases. Contrary to proteins and other RNA classes, miRs are stable and easily detectable in body fluids. In this respect, miRs represent a perfect candidate for minimal invasive biomarkers which can hopefully become a complement for invasive histological examinations of tumor tissue. Despite the high number of miR biomarker studies, the specificity and reproducibility of these studies is missing. Therefore, the standardization of pre‐analytical and analytical methods is urgently needed. Here, we validated miR analysis for RNA isolation and miR quantification by quantitative polymerase chain reaction (RT‐qPCR) based on good laboratory practice (GLP). Validation was carried out exemplarily on four miRs, which had already been described as potential biomarkers in previous studies. As basis for RNA analysis using RT‐qPCR, the Minimum Information for Publication of Quantitative Real‐Time PCR Experiments were applied and adapted on the analysis of circulating miRs from human plasma. In our study, we identified and solved several pitfalls from handling to normalization strategy in the analysis of extracellular miRs that lead to inconsistent and non‐repeatable data. Principles of GLP set a framework of experimental design, performance and monitoring to ensure high quality and reliable data. Within this study, we appointed first acceptance criteria for circulating miR quantification during validation which set standards for future miR quantification in blood samples.

MicroRNAs (miRs) are small noncoding RNAs which control the expression of target genes by either translational repression or RNA degradation, known as canonical miR functions. The recent discovery that miR-328 has a noncanonical function and can activate gene expression by antagonizing the activity of heterogeneous ribonuclear protein E2 (hnRNP E2) opens an unexplored and exciting field of gene expression regulation. The global importance of such noncanonical miR function is not yet known. In order to achieve a better understanding of the new miR activity, we performed a compartment specific tandem mass tag (TMT)-based proteomic analysis in differentiated MonoMac6 (MM6) cells, to monitor gene expression variations in response to miR-328 knockdown. We identified a broad spectrum of novel potential miR-328/hnRNP E2 and miR-328 targets involved in regulation of compartment specific cellular processes, such as inflammation or RNA splicing. This study provides first insights of the global significance of noncanonical miR function.

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