Background/Purpose:

Molecular mechanisms driving disease initiation and chronicity in RA are incompletely understood. There is increasing interest in the role played therein by microRNAs – small RNA species that mediate post-transcriptional regulation of integrated pathways in mammalian cells.  We recently profiled miRs in RA synovial fluid (SF) CD14+ macrophages in comparison to peripheral blood (PB) monocytes and identified dysregulation of miR-125a, which has not previously been associated with RA pathophysiology. Herein we aimed to characterise its expression and functional significance.

Methods:

Matched PB & SF CD14+ cells were isolated from RA patients (n=10). Additional PB samples were obtained from RA DMARD good responders (n=17), recurrent non-responders (n=11) and matched healthy controls (n=17). Primary human monocytes and THP-1 cells were stimulated with TLR ligands (LPS, PAM3, PolyIC, CLO97) and differentiated using M-CSF or GM-CSF for up to 7 days. Copy number of miR-125a was evaluated using qPCR. Targeted pathways were identified using prediction algorithms (e.g. TargetScan) and transcriptional profiling of SF CD14+ cells. Direct molecular interactions were confirmed using luciferase reporters. miR-125a or control mimic were transfected into THP-1 cells and IL-6R expression was evaluated by flow cytometry.

Results:

miR-125a was up-regulated in RA SF CD14+ macrophages compared to PB controls (p=0.002). Moreover, miR-125a was up-regulated in RA PB CD14+ monocytes compared with healthy controls, regardless of DMARD response status (p<0.02). Copy number of miR-125a in resting control PB monocytes was low. Extensive activation profiling revealed M-CSF, GM-CSF & 10% RA SF all induced miR125a expression at discrete time-points between 24h & 7ds. Of TLR ligands tested, only TLR4 agonism increased miR125a. Prediction algorithms identified members of the IL-6 signalling pathway (IL-6R, gp130) as potential targets of miR-125a. Luciferase reporter assays thereafter confirmed functional target interactions.  Transfection of THP-1 cells with miR-125a but not control mimic reduced IL-6R membrane expression measured by FACS. miR-125a also targeted the negative regulators of TLR signalling, TNFAIP3 and IRF4, assessed by TargetScan and luciferase reporter. Commensurate with this IRF4 was down-regulated in RA PB monocytes (both DMARD good & recurrent non-responders) compared to healthy donors.    

Conclusion:

Inflammatory cytokines, maturation factors and articular DAMPs drive elevated miR-125a expression in monocyte/macrophage lineages.  miR-125a, in turn represents a novel molecular pathway that cross regulates IL-6R and TLR pathway activation in RA macrophages. We conclude that miR-125a represents an intriguing molecular marker with therapeutic and biomarker potential.

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« Back to 2013 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/mir-125a-a-novel-regulator-of-il-6-and-tlr-driven-pathways-in-ra-pathogenesis/