Background/Purpose: Rheumatoid arthritis (RA) synovial fibroblasts (SF) are characterized by a stably activated phenotype which is, at least in part, because of the epigenetic inheritance of aberrations in gene expression. The histone methyltranferase EZH2 is upregulated in RASF thereby causing the epigenetic repression of specific target genes via methylation of histone 3 on lysine 27 (H3K27me3). Long noncoding RNAs (lncRNAs) have recently emerged as powerful regulators of gene expression, e.g. through the interaction with chromatin-associated proteins. In this regard, the lncRNA HOX transcript antisense RNA (HOTAIR) was found to associate with EZH2 and to regulate certain EZH2 target genes. Here we studied the function of HOTAIR in RASF.

Methods: SF were transfected using Lipofectamin 2000 (for siHOTAIR) or AMAXA Nucleofection (for plasmid DNA) and/or stimulated with 10ng/ml TNFα or 1ng/ml IL-1β. Gene expression was measured by quantitative real-time PCR (qPCR) and flow cytometry. Chromatin immunoprecipitation (ChIP) was performed with antibodies for histone 3 (H3), H3K27me3 or IgG control and precipitated chromatin was analyzed by qPCR. The activity of intracellular signaling pathways was determined by reporter gene assay (NF-κB; using the pGL4.32 vector and pRL_GAPDH as internal control) and Western blot (p38 phosphorylation).

Results: In OASF (n=13), the expression of HOTAIR was upregulated by 12.7-fold compared with RASF (n=9) (ΔCt 10.6±1.7 vs. 14.3±3.3, p=0.005). Stimulation of OASF (n=5) with TNFα or IL-1β potently decreased HOTAIR levels at 24h (by 65±8% and 50±9%) and 48h (by 52±31% and 49±27%) (p<0.05). ChIP analysis revealed increased levels of the repressive H3K27me3 mark at the HOTAIR promoter in RASF (ratio to H3: 0.51±0.35, OASF: 0.25±0.23; p=0.03) showing a strong inverse correlation with its expression (Spearman R=-0.8725, p<0.0001). Silencing of HOTAIR in OASF significantly increased the TNFα-induced activity of the NF-κB pathway (17±9-fold to 24±14-fold, n=13) whereas p38 phosphorylation was not significantly changed (n=8). In line with this, we found the expression of NF-κB target genes to be upregulated, namely intercellular adhesion molecule 1 (ICAM1) and vascular adhesion molecule 1 (VCAM1). At the mRNA level, ICAM1 and VCAM1 expression was increased after HOTAIR silencing (n=12) both under unstimulated (1.91±0.61- and 2.05±0.99-fold; p≤0.005) as well as TNFα-induced conditions (from 53±23- to 72±43-fold and 4.77±3.39- to 6.31±4.2-fold; p<0.005) whereas under IL-1β only ICAM1 mRNA levels were elevated (from 11.9±5.4- to 15.5±6.8-fold; p<0.005). These data were confirmed for ICAM1 protein levels measuring its surface expression under TNFα stimulation (n=6, p<0.01).

Conclusion: Our data underline the role of the epigenetically repressed lncRNA HOTAIR in the activated phenotype of RASF. Silencing of HOTAIR increased the activity of the NF-κB signaling pathway and the expression of adhesion molecules suggesting a contribution to inflammatory cell infiltration into the joint and, thus, chronic inflammation in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cytokine-mediated-repression-of-the-lncrna-hotair-enhances-intracellular-signaling-and-the-expression-of-adhesion-molecules-in-synovial-fibroblasts/