Veronika Hruskova

MicroRNAs (miRNAs) are small RNAs that regulate gene expression by targeting mRNA. It was proved that some miRNAs are significantly deregulated in rheumatoid arthritis (RA). MicroRNA-125b negatively regulates expression of TNF-α, which plays a crucial role in RA pathogenesis. The aim of this study was to determine the treatment outcome of patients with early RA based on the expression of circulating and cellular miR-125b. Total RNA was isolated from the plasma and peripheral blood mononuclear cells (PBMCs) of 58 patients with early RA before and three months after treatment initiation and of 54 age- and sex-matched healthy controls (HC). The expression of miR-125b was measured by TaqMan quantitative PCR. The treatment responders were defined as patients achieving remission or low disease activity (28-joint count disease activity score (DAS28) <3.2). Receiver operating characteristic (ROC) curve and stepwise backward multivariable logistic regression analyses of miR-125b expression were used to predict the disease outcome at three and six months after initiation of treatment. The expression of miR-125b in the PBMCs and plasma of treatment-naïve early RA patients was significantly lower than that of HC and increased significantly after three months of treatment, particularly in responders. However, only the cellular expression of miR-125b was inversely correlated with disease activity. MiR-125b expression in PBMCs was higher in responders than in non-responders after three months (p = 0.042). Using ROC analysis, the cellular expression of miR-125b, but not the disease activity at baseline, predicted the treatment response after three months of therapy (area under the curve 0.652 (95 % CI 0.510 to 0.793); p = 0.048). The expression of miR-125b in PBMCs of treatment-naïve patients may present a novel biomarker for monitoring the treatment outcome during the early phase of RA.

Background We have previously shown differential expression of some circulating miRNAs in patients with early RA (ERA) and their potential as markers of disease activity and treatment response. Objectives We aimed to investigate differentially expressed circulating miRNAs in clinically suspect arthralgia patients with positive antibodies to citrullinated peptide antigens (ACPA) who are at high risk of developing RA and compared them to ERA and established RA. Methods The study included 20 ACPA+ arthralgia patients, 25 treatment naïve ERA patients who met the 2010 ACR/EULAR criteria (disease duration Results Patients with arthralgia showed no clinical and ultrasound evidence of arthritis (grey scale and power Doppler for single joint ≤1) Of these, all were ACPA+, CRP 7.4±19.2 mg/l. Treatment naïve ERA patients had active disease (DAS28 5.7±1.3, CRP 20.7±23.2 mg/l), 60% were ACPA+. Patients with established RA all had high disease activity (DAS28 6.6±0.9, CRP 28.1±31.3 mg/l), 73% were ACPA+, had failed DMARDs therapy and were due to commence anti-TNF treatment. Out of the 380 miRNAs analysed by TLDA, 128 miRNAs were detected in HC, 129 in arthralgia, 44 in ERA and 25 in RA patients. TLDA analysis revealed 1.7x higher levels of miR-126 in ACPA+ arthralgia patients compared to HC, and 27x and 63x lower levels in ERA and RA compared to HC, respectively. These data were suggestive of significant dysregulation of miR-126 in patients with active polyarthritis compared to ACPA+ arthralgia. However, further validation showed no difference in miR-126 levels among HC, arthralgia and ERA patients but the levels were 2.6x lower in RA patients compared to HC (p=0.0037). The levels of miR-126 significantly negatively correlated with DAS28 (r=-0.460; p=0.047), SDAI (r=-0.535; p=0.018), SJC (r=-0.504; p=0.028), and TJC (r=-0.517; p=0.023) in treatment naïve ERA patients, but not in those with established disease. There was no correlation with CRP in any patient group. Conclusions Higher miR-126 levels reflect lower disease activity in treatment naïve ERA patients. We hypothesize that significant downregulation of miR-126 levels in RA patients may be modified by previous exposure to multiple treatments. Acknowledgement MHCR 023728 project Disclosure of Interest None declared

Background and objectives MicroRNAs (miRNAs) are small RNAs that regulate gene expression by targeting mRNA. It was proved that some miRNAs are significantly deregulated in rheumatoid arthritis (RA). MicroRNA-125b negatively regulates expression of TNF-α, which plays a crucial role in RA pathogenesis. The aim of this study was to evaluate the expression of miRNA-125b in peripheral blood mononuclear cells (PBMCs) and its association with disease activity and treatment response in early RA patients. Materials and methods A total of 58 (42 females; mean age 54.3 ± 16.4 years) early RA patients and 54 (41 females; mean age 50.9 ± 15.11 years) healthy controls (HC) were studied. Total RNA was isolated from PBMCs collected from patients before and after three months of glucocorticoid/disease modifying antirheumatic drugs treatment and from HC. The expression of miRNA-125b was determined by quantitative PCR. RNU44 was used for normalisation. Disease activity was defined using 28-Joint Count Disease activity Score (DAS28-ESR). The expression of miRNA-125b was studied in order to predict treatment response characterised by achieving remission or alternatively low disease activity (DAS28 < 3.2). Results The expression of miRNA-125b was significantly lower in early RA patients compared to HC (p = 0.001) and increased after three months of therapy (p = 0.006), mainly in responders (p = 0.019). At baseline, miRNA-125b expression negatively correlated with DAS28 (r = -0.462; p = 0.0003). In addition, baseline miRNA-125b expression predicted treatment response after three months which was performed by ROC curve analysis (AUC: 0.663 [95% CI 0.520 to 0.805]; p = 0.048). Conclusion Monitoring of miRNA-125b could be used as a biomarker of disease activity and treatment response in early RA. Acknowledgement IGA project No NT 14498; GAUK-367615.

Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFNγ. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFNγ treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFNγ or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.