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Abstract Number: 760

Antisense Long Noncoding RNA HAND2-AS1 and OTUD6B-AS1 Have Important Roles in the Pathogenesis of Systemic Sclerosis

Miki Takata1, Elena Pachera1, Anastasiia Kozlova1, Astrid Jüngel1, Tobias Messemaker2, Jeska de Vries-Bouwstra2, Tom W.J. Huizinga3, Fina Kurreeman2 and Oliver Distler4, 1Department of Rheumatology, Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland, Zurich, Switzerland, 2Department of Rheumatology, Leiden University Medical Center, Leiden, Netherlands, Leiden, Netherlands, 3Department of Rheumatology, LUMC, Leiden, Netherlands, Leiden, Netherlands, 4Department of Rheumatology, Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland

Meeting: ACR/ARHP Annual Meeting 2017

Date of first publication: September 18, 2017

Keywords:

Session Information

Date: Sunday, November 5, 2017

Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud's – Pathogenesis, Animal Models and Genetics Poster I

Session Type: ACR Poster Session A

Session Time: 9:00AM-11:00AM

Background/Purpose: Long noncoding RNAs (lncRNAs) represent a class of transcripts longer than 200 nucleotides that are not translated into proteins. In recent years, antisense (AS) lncRNAs have increasingly been recognized as important regulators of their sense genes and they have been found to play key roles in the pathogenesis of several diseases. However, the role of AS lncRNAs in SSc is still unknown. Our previous study identified two AS lncRNAs in SSc skin biopsies namely HAND2-AS1 and OTUD6B-AS1. Here we aim to characterize the functional relevance of these AS lncRNAs.

Methods: Sense and AS gene expression was analyzed by qPCR in RNA samples of HC and SSc dermal fibroblasts (Fb). Dermal Fb were stimulated with different profibrotic cytokines and growth factors like TGFβ, PDGF and IL-4 at physiological concentrations. Function of AS lncRNAs was analyzed by qPCR and Western Blot (WB) in HC dermal Fb and human pulmonary artery smooth muscle cells (HPASMC) transfected with locked nucleic acid antisense oligonucleotides (LNA GapmeRs).

Results: RNA sequencing analysis of skin biopsy revealed consistent and significant downregulation of HAND2-AS1 and OTUD6B-AS1 expression. In SSc and HC dermal Fb, no difference was recorded in the basal level of HAND2-AS1, HAND2, OTUD6B-AS1 and OTUD6B expression. However, HAND2 and HAND2-AS1 expression in SSc dermal Fb was significantly downregulated after TGFβ and IL-4 stimulation (n=4-5, p<0.05, up to 0.09 fold reduction). HAND2-AS1 expression was also significantly downregulated after PDGF stimulation (n=4, p<0.05, up to 0.12 fold reduction) and HAND2 expression had the same trend. OTUD6B expression in SSc dermal Fb was significantly downregulated after PDGF stimulation (n=7, p<0.05, up to 0.16 fold reduction) and OTUD6B-AS1 expression was also decreased. OTUD6B-AS1 expression was significantly downregulated after IL-4 stimulation (n=4, p<0.05, up to 0.52 fold reduction). Importantly, HAND2-AS1 knockdown significantly reduced collagen 1A1, fibronectin and α-smooth muscle actin (αSMA) mRNA in dermal Fb (p<0.05). Downregulation of fibronectin expression was also confirmed after HAND2-AS1 knockdown by WB analysis (n=4). In contrast to HAND2-AS1, OTUD6B-AS1 knockdown did not affect extracellular matrix production. However, we observed effects on cell cycle regulation after OTUD6B-AS1 knockdown. OTUD6B-AS1 knockdown in Fb and HPASMC significantly increased OTUD6B, and cell cycle regulators c-MYC, Cyclin D1 and Cyclin D2 mRNA (n=6, p<0.05). Upregulation of OTUD6B, c-MYC and Cyclin D1 in HPASMC was also confirmed after OTUD6B-AS1 knockdown by WB analysis (n=2).

Conclusion: This is the first report analyzing the functional role of AS lncRNAs in SSc. These results point to an important role of HAND2-AS1 in extracellular matrix production and of OTUD6B-AS1 in cell cycle progression.

Disclosure: M. Takata, None; E. Pachera, None; A. Kozlova, None; A. Jüngel, None; T. Messemaker, None; J. de Vries-Bouwstra, None; T. W. J. Huizinga, None; F. Kurreeman, None; O. Distler, Actelion, 5,Bayer, 5,Biogen Idec, 5,Boehringer Ingelheim, 5,ChemomAb, 5,espeRare Foundation, 5,Genentech/Roche, 5,GlaxoSmithKline, 5,Inventiva, 5,Lilly, 5,Medac, 5,MedImmune, 5,Mitsubishi Tanabe Pharma, 5,Pharmacyclics, 5,Novartis Pharmaceutical Corporation, 5,Pfizer Inc, 5,Sanofi, 5,Sinoxa, 5,UCB in the area of potential treatments of scleroderma and its complications, 5,Patent mir-29 for the treatment of systemic sclerosis licensed, 5,Actelion, 2,Bayer, 2,Biogen Idec, 2,Boehringer Ingelheim, 2,ChemomAb, 2,espeRare Foundation, 2,Genentech/Roche, 2,GlaxoSmithKline, 2,Inventiva, 2,Lilly, 2,Medac, 2,Medimmune, 2,Mitsubishi Tanabe Pharma, 2,Pharmacyclics, 2,Novartis, 2,Pfizer Inc, 2,Sanofi, 2,Sinoxa, 2,UCB in the area of potential treatments of scleroderma and its complications, 2,Patent mir-29 for the treatment of systemic sclerosis licensed, 2.

To cite this abstract in AMA style:

Takata M, Pachera E, Kozlova A, Jüngel A, Messemaker T, de Vries-Bouwstra J, Huizinga TWJ, Kurreeman F, Distler O. Antisense Long Noncoding RNA HAND2-AS1 and OTUD6B-AS1 Have Important Roles in the Pathogenesis of Systemic Sclerosis [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/antisense-long-noncoding-rna-hand2-as1-and-otud6b-as1-have-important-roles-in-the-pathogenesis-of-systemic-sclerosis/. Accessed .

ACR Meeting Abstracts - https://acrabstracts.org/abstract/antisense-long-noncoding-rna-hand2-as1-and-otud6b-as1-have-important-roles-in-the-pathogenesis-of-systemic-sclerosis/