Background/Purpose: Giant cell arteritis (GCA) is an inflammatory disease of large and medium-sized arteries. MiRNAs (miR) are small, non-coding RNAs that inhibit gene expression at post-transcriptional level. Several miRNAs have been reported as deregulated in temporal artery biopsies (TABs) from patients with GCA, but their role is still unknown. The present study aimed to investigate the effects of miR-146a and -146b on pathways relevant for GCA. These miRNAs have been found highly up-regulated in inflamed TABs from patients with GCA by our team and other two groups (Bolha L et al, Kuret T et al).

Methods: To identify upstream regulators of miR-146a and -146b expression, five primary cultures obtained from TABs were treated in vitro for 24 hours with cytokines known to be expressed in GCA lesions: IL-1β, IL-6, soluble IL-6R, IL-17, IL-22, IFNγ. Correlations between cytokine mRNA levels and miRNA levels were determined in inflamed TABs from 38 patients with GCA. To identify downstream effects of miR-146a and -146b, the primary cultures obtained from TABs and commercial human aortic endothelial and smooth muscle cells (HAoECs and HAoSMCs) were transfected with synthetic miR-146a and -146b to mimic miRNA activities. Results were compared with those obtained after transfection with a negative control oligonucleotide. Cell viability, miRNA and target gene expression, cytokine levels in culture supernatants were assayed after 6 days of treatment. Cell viability was determined by the WST-1 assay. MiRNA and gene expression were evaluated by real-time PCR. Cytokine levels in culture supernatants were measured by a multiplex bead-based assay.

Results: Treatment of primary cultures obtained from TABs with IL-1β and IL-17 increased miR-146a expression while treatment with IL-1β, IL-6 plus soluble IL-6R and IFNγ increased miR-146b expression. In inflamed TABs, mRNA levels of IFNγ correlated with levels of miR-146a (r=0.70, p< 0.0001) and mRNA levels of IL-1β correlated with levels of miR-146b (r=0.47, p=0.0032). Instead, no correlations were found between IL-6 or soluble IL-6R mRNAs and miR-146a or miR-146b levels. IL-17 gene expression was not detected. Transfection of miRNA mimics in cell cultures effectively increased miR-146a and -146b expression from 40 to 20000 fold. Following transfection, viability decreased in primary cultures from TABs (p< 0.05) but it did not change in HAoECs and HAoSMCs. Moreover, transfection of miR-146a and -146b mimics increased intercellular adhesion molecule-1 gene expression and production in soluble form by primary cultures from TABs (p< 0.05) but not by HAoECs and HAoSMCs. The negative control oligonucleotide did not have significant effects. Expression and/or production of IL-6, IL-8, vascular cell adhesion molecule, matrix metalloproteinase-2 (MMP-2), MMP-9, platelet-derived growth factor, epidermal growth factor, angiopoietin-1 were not affected.

Conclusion: Increased expression of miR-146a and -146b in GCA appeared to be driven by inflammatory cytokines (e.g. IL-1β, IFNγ) and to regulate viability and expression of adhesion molecules in cells from TABs. We thus hypothesize that miR-146a and -146b might modulate arterial microenvironment and leukocyte recruitment in GCA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-mir-146a-and-mir-146b-in-giant-cell-arteritis/