Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose
Analog to miRNAs that bind to argonaute proteins to suppress gene expression, a new group of non-coding RNAs piwi-interacting RNAs (piRNAs, 26-31 nt) build complexes with the argonaute-family members “P-element induced wimpy testis” (PIWI)-like proteins to repress retrotransposons in germ cells. In somatic cells a high expression of the PIWIL proteins was associated with cancer.
The aim of our study was to analyse the expression and regulation of PIWIL proteins in synovial cells and in peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA) focusing on the regulation of PIWIL4 by histone methylation and acetylation.
Methods
Expression of PIWIL mRNA was analysed by TaqMan RealTime-PCR and normalised to HPRT1 in synovial fibroblasts (SF) and synovial tissues (ST) from RA and osteoarthritis (OA) patients (n=5 each). PIWIL4 protein expression was analysed by Western blot.
RASF and OASF (n=5-12) were stimulated or not for 24 hours with Poly(I:C) (10ug/ml), LPS (100ng/ml) or TNFα (10ng/ml) in combination with IL1β (1ng/ml). RASF (n=3-6) were treated or not with the DNA methylation inhibitor 5-Azacytidine (5-AZA, 1uM, 7 days), the histone deacetylase inhibitor Trichostatin A (TSA, 1uM, 24h), the histone methylation inhibitor 3-Deazaneplanocin A (DZNep, 2.5uM, 48h) or with the methyl donor Betaine (50mM, 14 days).
After silencing PIWIL4 we assessed proliferation by cell counting. We analysed expression of PIWIL4 mRNA in PBMC isolated from patients with RA, systemic lupus erythematosus (SLE), axial spondyloarthritis (axSpA) and from healthy controls (HC, n=6-12 each).
Results
PIWIL2, 3 and 4 but not PIWIL1 could be detected in both RA and OASF without significant changes (RA/OA mean dCt=3.87/2.83, 9.50/12.32, 2.63/3.21). A high expression of PIWIL4 was also found in ST of patients with RA and OA (mean dCt=3.59/2.91).
Levels of PIWIL4 mRNA were further enhanced by Poly(I:C) in both RASF and OASF 2.9-fold (p=0.003)/3.4-fold (p=0.013); LPS 2.1-fold (p=0.026)/2.6-fold (p=0.025) and TNFα in combination with IL1β 1.9-fold (p=0.003)/1.7-fold (p=0.007). However, on the protein level no induction of PIWIL4 expression could be detected.
Treatment of RASF with 5-AZA did not regulate PIWIL4 expression, treatment with TSA down-regulated PIWIL4 mRNA to 0.4-fold (p=0.003); DZNep to 0.7-fold (p=0.026) and Betaine treatment caused 1.4-fold induction of PIWIL4 mRNA (p=0.043).
PIWIL4 silencing in RASF significantly decreased mRNA expression of the histone deacetylase HDAC1 (to 0.6-fold, p=0.0003), but not HDAC2 or HDAC3. Furthermore PIWIL4 silencing decreased cell proliferation in RASF stimulated with TNFα and IL1β by 30% (p=0.034).
In PBMC of patients with RA the expression of PIWIL4 was higher than in HC (mean dCt=2.18 vs. 2.64, p˂0.05), but lower than in patients with SLE (mean dCt=1.42, p˂0.001). There was no difference between RA and axSpA patients (mean dCt=2.30).
Conclusion
We have demonstrated that PIWIL4 expression is regulated by inflammatory cytokines and epigenetic modifications with functional consequences on proliferation suggesting a role of PIWIL4 in the activation of synovial fibroblasts in RA.
Disclosure:
L. Pleštilová,
FP7 OSTEOIMMUNE 289150,
2;
N. Gaur,
None;
M. Filková,
MHCR project 023728,
2;
B. Aradi-Vegh,
EuroTEAM,
2;
L. Senolt,
MHCR project 023728,
2;
A. Ciurea,
Pfizer Inc,
2,
Abbott Immunology Pharmaceuticals,
2,
Abbott Immunology Pharmaceuticals,
5,
Pfizer Inc,
5,
Merck Pharmaceuticals,
5,
UCB,
5;
R. E. Gay,
None;
J. Vencovsky,
MHCR project 023728,
2;
M. Neidhart,
None;
S. Gay,
None;
A. Juengel,
IMI-BTCure,
2,
IAR,
2.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/regulation-of-piwil4-by-histone-modifications-in-rheumatoid-arthritis/