OP0290 HOTAIR IS INVOLVED IN JOINT PATTERNING BY REGULATING FGFR2, BMP2 AND WNT PATHWAYS
Authors: Muriel Elhai, Mojca Frank-Bertoncelj, Kerstin Klein, Oliver Distler, Caroline Ospelt
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> Different forms of chronic arthritis such as rheumatoid arthritis (RA) or spondyloarthritis show a typical pattern of joint involvement. However, mechanisms involved in this patterning remain unknown. Synovial fibroblasts (SF) isolated from different joint regions, differ in their epigenetic landscape, their gene expression and their function. The long non-coding RNA HOTAIR, which is an important regulator of the epigenetic landscape, was found to be exclusively expressed in joints of the lower extremity. <h3>Objectives</h3> We aim to clarify the role of HOTAIR in SF. <h3>Methods</h3> HOTAIR was silenced in knee SF isolated from patients with osteoarthritis (OA) by HOTAIR GapmeR using Lipofectamine, and changes in gene expression were measured by next generation RNA sequencing (Illumina NovaSeq 6000) after 48h (n=3). Pathway analysis was performed using STRING protein network, and regulated genes were confirmed by real-time PCR in SF silenced for HOTAIR from OA and RA-patients (n=8) and compared with control SF transfected with Antisense LNA GapmeR negative control. The time course of targeted genes was studied with and without cycloheximide (10ug/ml 6h and 24h) (n=2). To assess the effect of HOTAIR silencing on the canonical Wnt pathway, we employed the TOP/FOP reporter system using the dual-luciferase kit (n=3). Regulation of HOTAIR and targeted genes expression was studied after stimulation with TNF-α (n=17), IL1, LPS, TLR2, TLR4 and poly I:C (n=5). The expression of HOTAIR and TNF-α were measured in synovial tissue from RA-patients (n=7) by qPCR. <h3>Results</h3> STRING analysis identified among others three significantly enriched pathways, namely FGF, BMP and Wnt, including 19 genes differentially expressed between SF silenced for HOTAIR and controls according to p-value (&lt;0.05) and log ratio (&gt;2.5 or &lt;-2.5). Real-time PCR confirmed 6 genes: FGFR2 (5 ± 0.1-fold downregulated; p&lt;0.01), FGF7 (1.9± 0.4-fold downregulated; p=0.02), BMP2 (1.7± 0.5-fold upregulated; p&lt;0.01), LGR5 (3.4±0.3-fold downregulated; p=0.02), CTNNB1 (beta-catenin) (1.5±0.5-fold upregulated; p&lt;0.01) and GSK3β (1.8± 0.2-fold downregulated; p=0.03). There was a trend for LRP6 (p=0.06). HOTAIR expression and FGFR2 and FGF7 expression were correlated (r= 0.67 and 0.55; p&lt;0.01) as well as HOTAIR and BMP2 (r=-0.45; p&lt;0.05). Time course of gene expression peaked after 30-48 hours. Adjunction of cycloheximide, blocking translation, did not change gene expression of BMP2 and CTNNB1 in SF silenced for HOTAIR, suggesting a direct effect of HOTAIR on these genes. FGFR2, FGF7, LGR5, LRP6 and GSK3β expression were normalized with cycloheximidine indicating the need of a protein to mediate HOTAIR effect on FGF pathway and on Wnt pathway. TopFlash assay confirmed the role of HOTAIR in regulation of Wnt pathway by 1.7± 0.2-fold decrease in Wnt activation after HOTAIR silencing (p=0.02). Stimulation by TNF-α led to a 2.1± 0.3 decrease in HOTAIR expression (p&lt;0.0001). Following TNF-α stimulation, FGFR2 and LGR5 expression decreased (p=0.0001 and 0.0004, respectively), whereas there was an increase in BMP2 expression (p&lt;0.0001), consistent with the effect of silencing HOTAIR on these genes. There was a decrease in HOTAIR expression following LPS (p=0.006), and poly I:C (p=0.003). HOTAIR and TNF-α were inversely correlated in the synovium of RA patients (r=-0.79; p&lt;0.05). <h3>Conclusion</h3> Our study highlights three main pathways regulated by HOTAIR and involved in joint patterning: FGFR2/FGF7, BMP2 and LRP6/LGR5/GSK3β/beta-catenin (Wnt). These pathways are known to play a role in normal joint development, but are also involved in inflammation, proliferation and apoptosis. Consistently, we observed that HOTAIR and targeted genes were selectively down-regulated by TNF-α suggesting a role of HOTAIR in the joint specific regulation of TNF-α mediated inflammatory arthritis. <h3>Disclosure of Interests</h3> Muriel ELHAI: None declared, Mojca Frank-Bertoncelj : None declared, Kerstin Klein: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Caroline Ospelt: None declared&nbsp;