Introduction: MicroRNAs (miRNAs), endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes, are present in human plasma in a stable form. In this study, we investigated whether miRNAs are also stably present in synovial fluids and whether plasma and synovial fluid miRNAs could be biomarkers of rheumatoid arthritis (RA) and osteoarthritis (OA). Methods: We measured concentrations of miR-16, miR-132, miR-146a, miR-155 and miR-223 in synovial fluid from patients with RA and OA, and those in plasma from RA, OA and healthy controls (HCs) by quantitative reverse transcription-polymerase chain reaction. Furthermore, miRNAs in the conditioned medium of synovial tissues, monolayer fibroblast-like synoviocytes, and mononuclear cells were examined. Correlations between miRNAs and biomarkers or disease activities of RA were statistically examined. Results: Synovial fluid miRNAs were present and as stable as plasma miRNAs for storage at -20°C and freeze-thawing from -20°C to 4°C. In RA and OA, synovial fluid concentrations of miR-16, miR-132, miR-146a, and miR-223 were significantly lower than their plasma concentrations, and there were no correlation between plasma and synovial fluid miRNAs. Interestingly, synovial tissues, fibroblast-like synoviocytes, and mononuclear cells secreted miRNAs in distinct patterns. The expression patterns of miRNAs in synovial fluid of OA were similar to miRNAs secreted by synovial tissues. Synovial fluid miRNAs of RA were likely to originate from synovial tissues and infiltrating cells. Plasma miR-132 of HC was significantly higher than that of RA or OA with high diagnosability. Synovial fluid concentrations of miR-16, miR-146a miR-155 and miR-223 of RA were significantly higher than those of OA. Plasma miRNAs or ratio of synovial fluid miRNAs to plasma miRNAs, including miR-16 and miR-146a, significantly correlated with tender joint counts and 28-joint Disease Activity Score. Conclusions: Plasma miRNAs had distinct patterns from synovial fluid miRNAs, which appeared to originate from synovial tissue. Plasma miR-132 well differentiated HCs from patients with RA or OA, while synovial fluid miRNAs differentiated RA and OA. Furthermore, plasma miRNAs correlated with the disease activities of RA. Thus, synovial fluid and plasma miRNAs have potential as diagnostic biomarkers for RA and OA and as a tool for the analysis of their pathogenesis.

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 (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. 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 Estimated Probability of RA by plasma MiRNA (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.

Osteoporosis is a metabolic bone disorder associated with compromised bone strength and an increased risk of fracture. Inhibition of the differentiation of bone-resorbing osteoclasts is an effective strategy for the treatment of osteoporosis. Prior work by our laboratory and others has shown that MYC promotes osteoclastogenesis in vitro, but the underlying mechanisms are not well understood. In addition, the in vivo importance of osteoclast-expressed MYC in physiological and pathological bone loss is not known. Here, we have demonstrated that deletion of Myc in osteoclasts increases bone mass and protects mice from ovariectomy-induced (OVX-induced) osteoporosis. Transcriptomic analysis revealed that MYC drives metabolic reprogramming during osteoclast differentiation and functions as a metabolic switch to an oxidative state. We identified a role for MYC action in the transcriptional induction of estrogen receptor–related receptor α (ERRα), a nuclear receptor that cooperates with the transcription factor nuclear factor of activated T cells, c1 (NFATc1) to drive osteoclastogenesis. Accordingly, pharmacological inhibition of ERRα attenuated OVX-induced bone loss in mice. Our findings highlight a MYC/ERRα pathway that contributes to physiological and pathological bone loss by integrating the MYC/ERRα axis to drive metabolic reprogramming during osteoclast differentiation.

Objective: MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate the activity of target messenger RNAs (mRNAs) and cellular processes. MicroRNA‐451 (miR‐451) is one of the miRNAs that is conserved perfectly among vertebrates, and it regulates cell proliferation, invasion, and apoptosis in tumors. However, the role of miR‐451 in autoimmune arthritis is unknown. This study was undertaken to identify the role of miR‐451 in autoimmune arthritis.Methods: We compared the expression of miR‐451 in neutrophils from patients with rheumatoid arthritis (RA) and healthy controls. We also evaluated the role of miR‐451 in neutrophil chemotaxis in vivo and in vitro using murine neutrophils. The regulation of p38 MAPK by miR‐451 was assessed. Double‐stranded miR‐451 was administered to SKG mice, the arthritis score was determined, and histologic examination was performed.Results: MicroRNA‐451 expression in neutrophils isolated from patients with RA was lower than that in healthy controls. Systemic administration of miR‐451 significantly disrupted the infiltration of neutrophils in an air‐pouch model of local inflammation without affecting apoptosis of neutrophils. Overexpression of miR‐451 significantly suppressed the migration of neutrophils to fMLP. We identified CPNE3 and Rab5a as direct targets of miR‐451. Overexpression of miR‐451 suppressed the phosphorylation of p38 MAPK via 14‐3‐3ζ, a known target of miR‐451, and Rab5a. In SKG mice, miR‐451 treatment reduced the severity of arthritis and the number of infiltrating cells.Conclusion: These results suggest that miR‐451 suppresses neutrophil chemotaxis via p38 MAPK and is a potential target in the treatment of RA.

In human inflammatory sites, PD-1^hiCXCR5⁻CD4⁺ T cells are involved in the formation of ectopic lymphoid-like structures (ELSs) by the secretion of chemokine CXCL13, but how the transcription of CXCL13 is regulated in CD4⁺ T cells is still unclear. Here we show that Sox4 is a key transcription factor for CXCL13 production in human CD4⁺ T cells under inflammatory conditions. In vitro TGF-β⁺, IL-2-neutralizing culture conditions give rise to PD-1^hiCXCR5⁻CD4⁺ T cells that preferentially express CXCL13, and transcriptome analysis and lentiviral overexpression indicate Sox4 association with the CXCL13 transcription. In vivo, Sox4 is significantly upregulated in synovial CD4⁺ T cells, when compared with blood CD4⁺ T cells, from patients with rheumatoid arthritis (RA), and further correlates with ELS formation in RA synovium. Overall, our studies suggest that Sox4 contributes to CXCL13 production and ELS formation at inflammatory sites in humans.

: MicroRNAs (miRNAs) are endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes. Although no miRNA has been reported to play an important role in the regulation of fracture healing, several miRNAs control key elements in tissue repair processes such as inflammation, hypoxia response, angiogenesis, stem cell differentiation, osteogenesis, and chondrogenesis. We compared the plasma concentrations of 134 miRNAs in 4 patients with trochanteric fractures and 4 healthy controls (HCs), and the levels of six miRNAs were dysregulated. Among these miRNAs, miR-92a levels were significantly decreased 24 hours after fracture, compared to HCs. In patients with a trochanteric fracture or a lumbar compression fracture, the plasma concentrations of miR-92a were lower on days 7 and 14, but had recovered on day 21 after the surgery or injury. To determine whether systemic downregulation of miR-92a can modulate fracture healing, we administered antimir-92a, designed using locked nucleic acid technology to inhibit miR-92a, to mice with a femoral fracture. Systemic administration of antimir-92a twice a week increased the callus volume and enhanced fracture healing. Enhancement of fracture healing was also observed after local administration of antimir-92a. Neovascularization was increased in mice treated with antimir-92a. These results suggest that plasma miR-92a plays a crucial role in bone fracture healing in human and that inhibition of miR-92a enhances fracture healing through angiogenesis and has therapeutic potential for bone repair. © 2014 American Society for Bone and Mineral Research.