Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
The Role of miR-155 in the Activation of RA and PsA Synovial Fluid Monocytes.
Kurowska-Stolarska2, Mariola S., Ballantine2, Lucy, Stolarski2, Bartosz E., Hunter2, John, Liew2, Foo Y., Gracie2, J. Alastair, McInnes1, Iain B.
MicroRNA (miR) network has emerged recently as an important post transcriptional regulator of immune response. Each individual miRNA species is capable of targeting a large number of distinct mRNA transcripts often belonging to one pathway. In our previous study we identified miR-155 to be strongly up-regulated in RA and PsA synovial fluid (SF) CD14+ cells compared to matched peripheral blood (PB) CD14+ cells suggesting that this miR may be involved in regulation of inflammatory pathways in arthritic joints. The aim of our study was to investigate the impact of miR 155 on the cytokine production and transcriptomic signature of RA and PsA SF monocyte and macrophage.
CD14+ cells from SF or PB of RA patients (n=14), PsA patients (n=13) and CD14+ cells from PB of healthy controls (n=6) were purified using CD14 MACS MicroBeads. PB CD14+ cells were transfected with miR-155 or scramble mimics (20 nM). Total RNA was isolated by miRNeasy kit. TaqMan miRNA and mRNA assays were used for semi-quantitative determination of the expression of miR-155 and its targets. The expressions of U6B small nuclear RNA or beta-actin were used as endogenous controls. Target prediction program and mRNA transcriptomic signature of RA and PsA SF CD14+ cells were employed to identify the cellular targets of miR-155 in SF CD14+ cells.
Overexpression of miR-155 in PB CD14+ monocytes and PB CD14+ derived macrophages triggered TNF production suggesting that miR-155 may indeed be involved in post-transcriptional control of inflammatory pathways in SF monocyte. A computational target ranking system (TargetScan human 5.1, aggregate Pct above 4.0) predicted 127 mRNA targets for miR-155. To identify mRNAs that are specifically targeted in SF monocytes we cross-referenced TargetScan predictions with a list of 2312 mRNAs that were differentially expressed in SF CD14+ cells compared to PB CD14+ cells of RA (n=8) and PsA (n=7) patients (GeneChip Affymetrix U133 plus 2). This approach produced a list of 26 miR-155 targets that were relevant to SF CD14+ activation. Among them, 24 genes were up-regulated and 2 genes were down-regulated in SF CD14+ cells compared to PB CD14+ cells. Since miRs exert their function by mRNA degradation, we focused on genes that were down-regulated in SF CD14+ cells, namely SHIP1 (Src homology-2 domain-containing inositol 5-phosphatase 1) and ZNF652 (zinc finger DNA-binding protein). Expression of SHIP1 was down-regulated by 9 and 3.9 fold in SF CD14+ cells from RA (n=8) and PsA (n=7) patients, respectively. ZNF652 was decreased by 4.43 and 2.97 fold in SF CD14+ cells from RA and PsA patients, respectively. The decrease of SHIP1 and ZNF652 expression in SF monocyte was confirmed by QPCR on additional patient samples. Experimental validation revealed that the expression of SHIP1 and ZNF652 was inhibited in PB CD14+ cells transfected with miR-155 mimic compared to control mimic transfected cells (48 ± 10% and 43± 7%, respectively).
This study identified functional miRNA-mRNA networks that may be responsible for pro-inflammatory activation of synovial fluid monocyte and macrophage in RA and PsA patients.
To cite this abstract, please use the following information:
Kurowska-Stolarska, Mariola S., Ballantine, Lucy, Stolarski, Bartosz E., Hunter, John, Liew, Foo Y., Gracie, J. Alastair, et al; The Role of miR-155 in the Activation of RA and PsA Synovial Fluid Monocytes. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1422