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OP0157 Apoptosis resistance of synovial fibroblasts of patients with rheumatoid arthritis is regulated by the long non-coding rna fas-as1
  1. A Pajak1,
  2. Y Horai2,
  3. E Pachera1,
  4. M Brock2,
  5. S Gay1,
  6. M Neidhart1,
  7. O Distler1,
  8. A Jungel1,
  9. on behalf of yes
  1. 1Department of Rheumatology
  2. 2Division of Pulmonology, University Hospital Zurich, Zurich, Switzerland

Abstract

Background Apoptosis resistance is thought to contribute to the accumulation of synoviocytes in the affected joints of patients with rheumatoid arthritis (RA). Of particular interest, the Fas receptor (FasR) - Fas ligand (FasL) apoptotic pathway appears altered in RA1. Long non-coding RNAs (lncRNAs) are emerging as key regulators of gene expression. Their role in disease, however, is still poorly understood. The recently described lncRNA FAS-AS1 has been implicated in alternative splicing of FasR. This results in increased amounts of soluble FasR (sFasR) and thereby prevents FasL-induced cell death2,3. Whether lncRNA FAS-AS1 is involved in the apoptosis resistance of synovial fibroblasts in RA is unknown.

Objectives To assess the regulatory role of lncRNA FAS-AS1 in the apoptosis resistance of synovial fibroblasts from patients with RA (RASF).

Methods Levels of expression of lncRNA FAS-AS1 were measured in RASF and synovial fibroblasts from patients with osteoarthritis (OASF) by qPCR using SYBRGreen detection. Cells were treated with TNFα (10ng/ml, 24h) and/or with FasL (50ng/ml, 18h) to assess the secreted amount of sFasR by ELISA and the induction of apoptosis by Annexin V staining followed by flow cytometry. LncRNA FAS-AS1 was silenced using locked nucleic acid antisense oligonucleotides (GapmeR).

Results There was no significant difference in basal levels of lncRNA FAS-AS1 expression between RASF and OASF (n=4 each). TNFα stimulation of synovial fibroblasts, regardless of the disease context (RA or OA), resulted in higher than 6-fold induction of lncRNA FAS-AS1 expression (6.45±1.39; p<0,05; n=4 for RASF and 6.26±1.47; p=0.05; n=4 for OASF). In addition, TNFα stimulation induced secretion of sFasR in RASF from 107±74 to 390±274pg/ml (p<0.05; n=6) and OASF from 69±54 to 249±134pg/ml (p<0.05; n=6). FasL induced apoptosis in both RASF and OASF (55–75±13%). However, pretreatment with TNFα reduced the FasL-induced apoptosis in RASF by 25±19% and in OASF by 15±10%. Silencing with GapmeR successfully decreased the expression of lncRNA FAS-AS1 by 40±22% SEM. Most interestingly, silencing of lncRNA FAS-AS1 reverted the TNFα inhibitory effect on FasL-induced apoptosis by 37±11% (n=4).

Conclusions Our data revealed a novel mechanism, which may underlie apoptosis resistance in RASF. We showed that in a pro-inflammatory cytokine milieu, lncRNA FAS-AS1 up-regulates the release of sFasR and thereby may lower the responsiveness of cells to death signals. Thus, targeting lncRNA FAS-AS1 might prevent apoptosis resistance and synovial hyperplasia in RA.

References

  1. Hong et al., Life Sciences. 2015; Feb 1; 122:37–41.

  2. Seghal et al., Leukemia. 2014; Dec 28(12); 2376–87.

  3. Villamizar et al., Oncotarget. 2016; Mar 22; 7(12):13810–26.

References

Disclosure of Interest A. Pajak: None declared, Y. Horai: None declared, E. Pachera: None declared, M. Brock: None declared, S. Gay Grant/research support from: EU project BTCure, IAR, Consultant for: GSK, M. Neidhart Grant/research support from: Baugartenstiftung, O. Distler Grant/research support from: Actelion, Bayer, Boehringer Ingelheim, Pfizer, Sanofi, Consultant for: 4 D Science, Actelion, Active Biotec, Bayer, BiogenIdec, BMS, Boehringer Ingelheim, ChemomAb, EpiPharm, espeRare foundation, Genentech/Roche, GSK, Inventiva, Lilly, medac, Mepha, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Pfizer, Sanofi, Serodapharm, Sinoxa, Speakers bureau: AbbVie, iQone Healthcare, Mepha, A. Jungel: None declared

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