Identification of non-coding RNAs controlling gout-relevant genes
Nils Asmann (1), Nicholas A. Sumpter (1), Brenda Kischkel (1), Ezio T. Fok (2,3), Mumin Ozturk (2,3), Riku Takei (4), Megan P. Leask (6), Musa M. Mhlanga (2,3), Tony R. Merriman (4,5), Leo A. B. Joosten (1,7)
Affiliation(s):
1. Radboud University Medical Center, Department of Internal Medicine, 6500HB, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands. Phone: +31-06-84452806, mail: Nils.Asmann@radboudumc.nl
2. Department of Cell Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, the Netherlands
3. Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands
4. Division of Clinical Immunology and Rheumatology, University of Alabama Birmingham, Birmingham, Alabama, USA
5. Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
6. Department of Physiology, University of Otago, Dunedin, New Zealand
7. Iuliu Hatieganu University of Medicine and Pharmacy, Department of Medical Genetics, Cluj-Napoca, Romania
Background: Gout is characterized by flares caused by monosodium urate (MSU) crystal deposition and subsequent activation of innate immune cells. A recent genome-wide association study (GWAS) identified many gout-associated variants that may affect genes related to this immune response. It is important to translate these genetic associations into mechanistic and molecular insights for new druggable targets to prevent gout flares. Hereby, we focus on non-coding RNAs (ncRNAs) controlling the transcription levels of putative gout flare-related genes (Fig. 1) that are not associated with hyperuricemia. This includes the previously unknown CSF1-CSF1R axis in gout.
Objectives: In this study, we aimed to investigate a potential immune gene-priming long non-coding RNA (IPL) and enhancer RNA (eRNA) of colony-stimulating factor 1 (CSF1) and assessed its role in gouty arthritis.
Methods: Characterization was done on the transcription levels (IPL, eRNA, and mRNA) in a control and stimulated setup to validate the presence of these RNAs and their effects on CSF1 concentrations. Initial experiments were done in THP-1 cells to establish the technology needed for the extraordinarily low expressed RNAs. Based on the GWAS hits, we focused to validate the IPL, eRNA, and CSF1-mRNA in qPCR and dPCR experiments. Furthermore, the Olink proximity extension assay was used to determine CSF1 plasma concentrations in gout patients within a flare or the inter-critical phase.
Findings: In order to study specifically CSF1 as well as its potential regulatory ncRNAs, we designed an experimental setup upregulating CSF1 expression. Initial experiments were carried out with 4 to 96 hours PMA-stimulated THP-1 cells, since they had higher availability and higher RNA concentrations. This allowed us to verify upregulation of CSF1 and CSF1R by PMA and the link between the eRNA and IPL transcription. Whereby CSF1 and CSF1R show a continuous increase over the time course, the ncRNAs suggest an initial peak after 4h stimulation, thus suggesting a different order of activation by PMA. Future experiments will be focussed on primary human monocytes of gout patients, resembling in vivo settings.
Significance: This study improves the understanding of the transition from hyperuricemia to gout by focusing on gout-associated genes that do not appear to play a role in hyperuricemia. Furthermore, unravelling the molecular pathways will improve therapeutical interventions by providing novel targets for gout.
