OBJECTIVE: The PIWIL (P-element induced wimpy testis like protein) subfamily of argonaute proteins is essential for Piwi-interacting RNA (piRNA) biogenesis and their function to silence transposons during germ-line development. Here we explored their presence and regulation in rheumatoid arthritis (RA). METHODS: The expression of PIWIL genes in RA and osteoarthritis (OA) synovial tissues and synovial fibroblasts (SF) was analysed by Real-time PCR, immunofluorescence and Western blot. The expression of piRNAs was quantified by next generation small RNA sequencing (NGS). The regulation of PIWI/piRNAs, proliferation and methylation of LINE-1 after silencing of PIWIL genes were studied. RESULTS: PIWIL2 and 4 mRNA were similarly expressed in synovial tissues and SF from RA and OA patients. However, on the protein level only PIWIL4 was strongly expressed in SF. Using NGS up to 300 piRNAs were identified in all SF without significant differences in expression levels between RA and OASF. Of interest, the analysis of the co-expression of the detected piRNAs revealed a less tightly regulated pattern of piRNA-823, -4153 and -16659 expression in RASF. In RASF and OASF, stimulation with TNFα+IL1β/TLR-ligands further significantly increased the expression levels of PIWIL2 and 4 mRNA and piRNA-16659 was significantly (4-fold) induced upon Poly(I:C) stimulation. Silencing of PIWIL2/4 neither affect LINE-1 methylation/expression nor proliferation of RASF. CONCLUSION: We detected a new class of small regulatory RNAs (piRNAs) and their specific binding partners (PIWIL2/4) in synovial fibroblasts. The differential regulation of co-expression of piRNAs in RASF and the induction of piRNA/Piwi-proteins by innate immune stimulators suggest a role in inflammatory processes.

Center of Experimental Rheumatology University Hospital Zürich Zürich Switzerland

Department of Rheumatology University Hospital Zürich Zürich Switzerland

Functional Genomic Centre Zürich Zürich Switzerland

Institute of Rheumatology and Clinic of Rheumatology 1st Faculty of Medicine Charles University Prague Prague Czech Republic

Schulthess Clinic Zürich Switzerland

See more in PubMed

Klareskog L, Catrina AI, Paget S. Rheumatoid arthritis. Lancet. 2009;373(9664):659–72. Epub 2009/01/23. 10.1016/S0140-6736(09)60008-8 PubMed DOI

Neumann E, Lefevre S, Zimmermann B, Gay S, Muller-Ladner U. Rheumatoid arthritis progression mediated by activated synovial fibroblasts. Trends Mol Med. 2010;16(10):458–68. Epub 2010/08/27. 10.1016/j.molmed.2010.07.004 PubMed DOI

Neidhart M, Rethage J, Kuchen S, Kunzler P, Crowl RM, Billingham ME, et al. Retrotransposable L1 elements expressed in rheumatoid arthritis synovial tissue: association with genomic DNA hypomethylation and influence on gene expression. Arthritis and rheumatism. 2000;43(12):2634–47. Epub 2001/01/06. 10.1002/1529-0131(200012)43:12<2634::AID-ANR3>3.0.CO;2-1 PubMed DOI

Karouzakis E, Gay RE, Michel BA, Gay S, Neidhart M. DNA hypomethylation in rheumatoid arthritis synovial fibroblasts. Arthritis and rheumatism. 2009;60(12):3613–22. Epub 2009/12/02. 10.1002/art.25018 PubMed DOI

Sai Lakshmi S, Agrawal S. piRNABank: a web resource on classified and clustered Piwi-interacting RNAs. Nucleic acids research. 2008;36(Database issue):D173–7. Epub 2007/09/21. PubMed Central PMCID: PMCPmc2238943. 10.1093/nar/gkm696 PubMed DOI PMC

Bamezai S, Rawat VP, Buske C. Concise review: The Piwi-piRNA axis: pivotal beyond transposon silencing. Stem cells (Dayton, Ohio). 2012;30(12):2603–11. Epub 2012/09/22. PubMed

Ng KW, Anderson C, Marshall EA, Minatel BC, Enfield KS, Saprunoff HL, et al. Piwi-interacting RNAs in cancer: emerging functions and clinical utility. Mol Cancer. 2016;15(1):5. Epub 2016/01/16. PubMed Central PMCID: PMCPMC4714483. PubMed PMC

Greither T, Koser F, Kappler M, Bache M, Lautenschlager C, Gobel S, et al. Expression of human Piwi-like genes is associated with prognosis for soft tissue sarcoma patients. BMC cancer. 2012;12:272 Epub 2012/07/04. PubMed Central PMCID: PMCPmc3472179. 10.1186/1471-2407-12-272 PubMed DOI PMC

Chen C, Liu J, Xu G. Overexpression of PIWI proteins in human stage III epithelial ovarian cancer with lymph node metastasis. Cancer biomarkers: section A of Disease markers. 2013;13(5):315–21. Epub 2014/01/21. 10.3233/CBM-130360 PubMed DOI

Cui L, Lou Y, Zhang X, Zhou H, Deng H, Song H, et al. Detection of circulating tumor cells in peripheral blood from patients with gastric cancer using piRNAs as markers. Clinical biochemistry. 2011;44(13):1050–7. Epub 2011/06/28. 10.1016/j.clinbiochem.2011.06.004 PubMed DOI

Su C, Ren ZJ, Wang F, Liu M, Li X, Tang H. PIWIL4 regulates cervical cancer cell line growth and is involved in down-regulating the expression of p14ARF and p53. FEBS letters. 2012;586(9):1356–62. Epub 2012/04/10. 10.1016/j.febslet.2012.03.053 PubMed DOI

Cheng J, Deng H, Xiao B, Zhou H, Zhou F, Shen Z, et al. piR-823, a novel non-coding small RNA, demonstrates in vitro and in vivo tumor suppressive activity in human gastric cancer cells. Cancer letters. 2012;315(1):12–7. Epub 2011/11/04. 10.1016/j.canlet.2011.10.004 PubMed DOI

He X, Chen X, Zhang X, Duan X, Pan T, Hu Q, et al. An Lnc RNA (GAS5)/SnoRNA-derived piRNA induces activation of TRAIL gene by site-specifically recruiting MLL/COMPASS-like complexes. Nucleic acids research. 2015;43(7):3712–25. Epub 2015/03/18. PubMed Central PMCID: PMCPMC4402533. 10.1093/nar/gkv214 PubMed DOI PMC

Ross RJ, Weiner MM, Lin H. PIWI proteins and PIWI-interacting RNAs in the soma. Nature. 2014;505(7483):353–9. PubMed Central PMCID: PMC4265809. 10.1038/nature12987 PubMed DOI PMC

Rajasethupathy P, Antonov I, Sheridan R, Frey S, Sander C, Tuschl T, et al. A role for neuronal piRNAs in the epigenetic control of memory-related synaptic plasticity. Cell. 2012;149(3):693–707. Epub 2012/05/01. PubMed Central PMCID: PMCPmc3442366. 10.1016/j.cell.2012.02.057 PubMed DOI PMC

Zhong F, Zhou N, Wu K, Guo Y, Tan W, Zhang H, et al. A SnoRNA-derived piRNA interacts with human interleukin-4 pre-mRNA and induces its decay in nuclear exosomes. Nucleic acids research. 2015;43(21):10474–91. Epub 2015/09/26. PubMed Central PMCID: PMCPMC4666397. 10.1093/nar/gkv954 PubMed DOI PMC

Peng L, Song L, Liu C, Lv X, Li X, Jie J, et al. piR-55490 inhibits the growth of lung carcinoma by suppressing mTOR signaling. Tumour Biol. 2015. Epub 2015/09/27. PubMed

Filkova M, Jungel A, Gay RE, Gay S. MicroRNAs in rheumatoid arthritis: potential role in diagnosis and therapy. BioDrugs. 2012;26(3):131–41. Epub 2012/04/13. 10.2165/11631480-000000000-00000 PubMed DOI

Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis and rheumatism. 1988;31(3):315–24. Epub 1988/03/01. PubMed

Aradi B, Kato M, Filkova M, Karouzakis E, Klein K, Scharl M, et al. Protein Tyrosine Phosphatase Nonreceptor Type 2 (PTPN2), an Important Regulator of IL-6 Production in Rheumatoid Arthritis Synovial Fibroblasts. Arthritis & rheumatology (Hoboken, NJ). 2015. Epub 2015/07/04. PubMed

Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome biology. 2009;10(3):R25 Epub 2009/03/06. PubMed Central PMCID: PMCPMC2690996. 10.1186/gb-2009-10-3-r25 PubMed DOI PMC

Varet H, Coppée J-Y, Dillies M-A. SARTools: a DESeq2- and edgeR-based R pipeline for comprehensive differential analysis of RNA-Seq data2015 2015-01-01 00:00:00. PubMed PMC

Sugimoto K, Kage H, Aki N, Sano A, Kitagawa H, Nagase T, et al. The induction of H3K9 methylation by PIWIL4 at the p16Ink4a locus. Biochemical and biophysical research communications. 2007;359(3):497–502. Epub 2007/06/05. 10.1016/j.bbrc.2007.05.136 PubMed DOI

Martinez VD, Vucic EA, Thu KL, Hubaux R, Enfield KS, Pikor LA, et al. Unique somatic and malignant expression patterns implicate PIWI-interacting RNAs in cancer-type specific biology. Scientific reports. 2015;5:10423 Epub 2015/05/28. PubMed Central PMCID: PMCPMC4444957. 10.1038/srep10423 PubMed DOI PMC

Kang HJ, Moon MJ, Lee HY, Han SW. Testosterone alters testis function through regulation of piRNA expression in rats. Molecular biology reports. 2014;41(10):6729–35. Epub 2014/07/07. 10.1007/s11033-014-3558-y PubMed DOI

Pan Y, Hu M, Liang H, Wang JJ, Tang LJ. The expression of the PIWI family members miwi and mili in mice testis is negatively affected by estrogen. Cell Tissue Res. 2012;350(1):177–81. Epub 2012/05/26. 10.1007/s00441-012-1447-z PubMed DOI

Neidhart M, Karouzakis E, Schumann GG, Gay RE, Gay S. Trex-1 deficiency in rheumatoid arthritis synovial fibroblasts. Arthritis and rheumatism. 2010;62(9):2673–9. Epub 2010/05/25. 10.1002/art.27567 PubMed DOI