The Long Noncoding RNA HOTTIP Regulates Cell Cycle and Inflammatory Response in Hand Synovial Fibroblasts

Mojca Frank-Bertoncelj ¹, Tadeja Kuret², Ana Županič ³, Snezna Sodin-Semrl ⁴, Oliver Distler ⁵ and Caroline Ospelt ¹, ¹University Hospital Zürich, Zürich, Switzerland, ²University Hospital Zürich, Zurich, Tajikistan, ³University Hospital Zürich, Zurich, Switzerland, ⁴University Medical Center Ljubljana, Ljubljana, Slovenia, ⁵Dept. of Rheumatology, University Hospital Zürich, Zürich, Switzerland, Zürich, Switzerland

Meeting: 2019 ACR/ARP Annual Meeting

Keywords: Inflammation, matrix metalloproteinase (MMP) and chemokines, Rheumatoid arthritis (RA), synovial cells, synovial fluid

Session Information

Date: Sunday, November 10, 2019

Title: 3S079: RA – Etiology & Pathogenesis I (815–820)

Session Type: ACR Abstract Session

Session Time: 2:30PM-4:00PM

Background/Purpose: Previously we showed that the long noncoding RNA (lncRNA) HOTTIP is exclusively expressed in synovial fibroblasts (SF) from distal joints, such as small joints of hands and feet, which exhibit prominent proliferative and chemotactic activities (Frank-Bertoncelj, et al. Nat Commun, 2017). Our objective was to explore the role of HOTTIP in shaping the function of hand SF in arthritis.

Methods: We silenced the lncRNA HOTTIP in hand SF using LNA GapmeRs (100 nM, 48h). We conducted RNA-sequencing (n=2) and confirmed RNA-sequencing data with qPCR in a larger cohort of hand SF from RA patients (n=6). Protein-protein interaction analysis of RNA-sequencing data was performed using STRING. Protein levels of IL8 (n=4) and MMP3 (n=6) in cell supernatants were measured by ELISA. Cyclin dependent kinase inhibitor p21 (n=4) was detected by Western blot. Proliferation (n=3) was measured in vitro using the BrdU assay. Apoptosis and necrosis (n=6) were determined with Real time-Glo annexin V apoptosis and necrosis assay. Significance was defined as p< 0.05 measured by one-sample t-test or paired t-test.

Results: STRING analysis of RNA sequencing data showed changes in cell cycle, inflammatory response and integrin pathways after silencing of HOTTIP in hand SF. We confirmed the significant downregulation of transcripts involved in mitotic cell cycle (NCAPG, TUBGCP5, TADA3, ASPM, ZWILCH, CDC27, BUB1, GPSM2 and CDK6), significantly increased transcript and protein expression of the cyclin-dependent kinase inhibitor p21 and significantly less proliferation in HOTTIP silenced hand SF. No difference in apoptosis and necrosis was observed between HOTTIP and control silenced hand SF. Furthermore, silencing of HOTTIP resulted in significant upregulation of transcripts involved in inflammatory and immune response pathways (IL8, IL12A, IL17C, CXCL3, MMP3 and TNFAIP3) and significantly increased protein levels of IL8 and MMP3. Silencing of HOTTIP also significantly altered gene expression of different types of integrins (ITGA3, ITGB1, ITGB5, ITGB7 and ITGA2B) that play a role in adhesion and organization of newly synthesized extracellular matrix. Stimulation of hand SF with TNFα and IL6/IL6R resulted in significant decrease of HOTTIP expression.

Conclusion: Distal-specific expression of HOTTIP could support enhanced proliferative properties in hand SF. In inflammatory conditions, reduced levels of HOTTIP might shape a location-specific inflammatory response with joint-specific changes in cytokine and chemokine expression, cell adhesion and cell-to-extracellular matrix interactions.

Disclosure: M. Frank-Bertoncelj, Kurt und Senta Herrmann Foundation, 2, Promedica Foundation, 2; T. Kuret, EULAR Scientific Bursary, 2, IUBMB Wood-Whelan Fellowship, 2, Swiss-European Mobility Programme (SEMP) Scholarship, 2, Slovenian Research Agency, 2; A. Županič, EULAR Scientific Bursary, 2; S. Sodin-Semrl, None; O. Distler, A. Menarini, 5, Abbvie, Acceleron, 5, Acceleron Pharma, 5, Actelion, 2, 5, 8, Actelion Pharmaceuticals, 2, 5, 8, 9, Amgen, 5, AnaMar, 2, 5, Bayer, 2, 5, 8, 9, Biogen Idec, 2, 5, Blade Therapeutics, 5, Boehringer Ingelheim, 2, 5, 8, 9, Catenion, 5, 9, ChemomAb, 2, 5, ChemomAB, 5, CSL Behring, 5, Ergonex, 5, espeRare Foundation, 2, 5, Genentech/Roche, 2, 5, GlaxoSmithKline, 5, GSK, 2, 5, Holds Patent mir-29 for the treatment of systemic sclerosis, 9, Inventiva, 2, 5, iQvia, 5, Italfarmaco, 2, 5, Italfarmco, 5, Lilly, 2, 5, med, 5, 8, medac, 5, Medac, 2, 5, MedImmune, 2, 5, Medscape, 5, 8, 9, Menarini, 8, Mepha, 8, Mitsubishi Tanabe, 2, 5, Mitsubishi Tanabe Pharma, 2, 5, MSD, 5, 8, Novartis, 2, 5, 8, 9, Patent, 9, Patent issued, 9, Pfizer, 2, 5, 8, Pharmacyclics, 2, 5, Roche, 5, 8, 9, Sanofi, 2, 5, Sinoxa, 2, 5, Target Bio Science, 5, Target BioScience, 5, UCB, 2, 5, 9, UCB in the area of potential treatments of scleroderma and its complications, 2, 5; C. Ospelt, Kurt und Senta Herrmann Foundation, 2, Promedica Foundation, 2.

To cite this abstract in AMA style:

Frank-Bertoncelj M, Kuret T, Županič A, Sodin-Semrl S, Distler O, Ospelt C. The Long Noncoding RNA HOTTIP Regulates Cell Cycle and Inflammatory Response in Hand Synovial Fibroblasts [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/the-long-noncoding-rna-hottip-regulates-cell-cycle-and-inflammatory-response-in-hand-synovial-fibroblasts/. Accessed .

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