Session Title: Cytokines, Mediators, and Gene Regulation
Session Type: Abstract Submissions (ACR)
A recent genome wide association study identified the variant rs26232 in the first exon of an uncharacterized gene C5orf30. In addition, this variant is also associated with severity of radiological joint damage suggesting a role in tissue breakdown. To date there is no function assigned for C5orf30 and neither the gene or protein coded show homology to any known functional sequences. However, C5orf30 is highly conserved in chimpanzee, dog, cow, mouse, chicken, and zebrafish (orthologs). The aim of this study is to determine the biological roles of C5orf30 in health and RA.
Real time PCR and western blotting was used to determine C5orf30 transcript and protein levels in fibroblast-like synovial cells (FLS-stimulated with TNF & hypoxia) and peripheral blood leukocytes isolated from RA patients and healthy individuals. Immunohistochemistry using synovial samples was used to determine cellular expression using anti-C5orf30 and antibodies to macrophages (CD68), fibroblasts (5B5), T (CD3) & B (CD19) cells. To investigate gene function siRNA was used to knockdown C5orf30 in synovial FLS in vitro and we have used morpholino antisense oligonucleotide (MO)-mediated knocked down of C5orf30 in zebrafish embryos (fms:mcherry) in vivo.
Expression of C5orf30 was detected at lower levels in peripheral blood leukocytes of RA patients compared to healthy controls (117 patients vs. 102 controls, p=0.00052). C5orf30 was expressed in joint FLS and was found to be up regulated following treatment with hypoxia (8-fold) and down-regulated by TNF-a (0.5-fold). Confocal microscopy revealed C5orf30 was strongly expressed in both the nuclear and cytoplasmic compartment of synovial lining cells including macrophages and fibroblasts but not B & T cells. C5orf30 was undetectable in arthroscopy sections obtained from osteoarthritis or control (knee replacement) patients. So far, 80% C5orf30 knockdown has been achieved in FLS without affecting cell viability. Interestingly, MO-mediated knockdown of C5orf30 impeded zebrafish development and increased total macrophage numbers by 40% compared to knockdown of a scrambled control MO.
C5orf30 is expressed at both the transcript & protein level in synovial cells but not in circulating PBMC obtained from RA patients, suggesting that C5orf30 is expressed in a tissue-specific manner. We are currently assessing the morphological phenotype and function arising from C5orf30 siRNA knockdown in FLS using confocal microscopy and matrigel invasion assays. NMR and mass spectrometry experiments are on the way to determine the three-dimensional structure of C5orf30 and its potential protein-binding partners.
G. A. Adeleke,
A. G. Wilson,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/biological-roles-of-c5orf30-in-rheumatoid-arthritis/