• Corpus ID: 45990061

articleAltered microRNA expression profile with miR-146 a upregulation in CD 4 + T cells from patients with rheumatoid arthritis

@inproceedings{Li2015articleAlteredME,
  title={articleAltered microRNA expression profile with miR-146 a upregulation in CD 4 + T cells from patients with rheumatoid arthritis},
  author={Jingyi Li and Ying Wan and Qiuye Guo and Liyun Zou and Jinyu Zhang and Yongfei Fang and Jingbo Zhang and Jinjun Zhang and Xiao-lan Fu and Hongli Liu and Liwei Lu and Yuzhang Wu},
  year={2015}
}

• Jingyi Li, Y. Wan, +9 authors Yuzhang Wu
• Published 2015
• Biology, Medicine

Introduction: Increasing evidence indicates that microRNAs (miRNAs) play a critical role in the pathogenesis of inflammatory diseases. The aim of the study was to investigate the expression pattern and function of miRNAs in CD4+ T cells from patients with rheumatoid arthritis (RA). Methods: The expression profile of miRNAs in CD4+ T cells from synovial fluid (SF) and peripheral blood of 33 RA patients was determined by microarray assay and validated by qRT-PCR analysis. The correlation between altered expression of miRNAs and cytokine levels was determined by linear regression analysis. The role of miR-146a overexpression in regulating T cell apoptosis was evaluated by flow cytometry. A genome-wide gene expression analysis was further performed to identify miR-146a-regulated genes in T cells. Results: miRNA expression profile analysis revealed that miR-146a expression was significantly upregulated while miR363 and miR-498 were downregulated in CD4+ T cells of RA patients. The level of miR-146a expression was positively correlated with levels of tumor necrosis factor-alpha (TNF-α), and in vitro studies showed TNF-α upregulated miR-146a expression in T cells. Moreover, miR-146a overexpression was found to suppress Jurkat T cell apoptosis. Finally, transcriptome analysis of miR-146a overexpression in T cells identified Fas associated factor 1 (FAF1) as a miR-146aregulated gene, which was critically involved in modulating T cell apoptosis. Conclusions: We have detected increased miR-146a in CD4+ T cells of RA patients and its close correlation with TNF-α levels. Our findings that miR-146a overexpression suppresses T cell apoptosis indicate a role of miR-146a in RA pathogenesis and provide potential novel therapeutic targets. Introduction Rheumatoid arthritis (RA) is a common chronic inflammatory disease characterized by radiographic joint destruction with severe functional deterioration and high mortality. A hallmark pathological feature of RA is the infiltration and accumulation of T cells in the synovium of joint [1]. As the shared epitope in human leukocyte antigen-DR genes is found in about 80% of RA patients, dysregulated CD4+ T cell activation and function have been investigated based on the available evidence of genetic predisposition [2,3]. T cells isolated from joint tissue and synovial fluid (SF) show an activated and memory phenotype and appear to respond poorly to stimulation with mitogen or antigens in vitro [4,5]. These T cells are unusually resistant to apoptosis in SF that contains a significant amount of proapoptotic factors such as bioactive FasL, TRAIL and TNF-α [6,7]. In addition, studies on a murine model of proteoglycan-induced arthritis also showed that CD4+ T cells failed to undergo apoptosis [8]. All these findings from patients and animal models suggest that the inhibition of T cell death may result in the persistence and accumulation of T cells in synovium, as well as the accumulation of T cells in the periphery. The long-term sur* Correspondence: [e-mail] 1 Institute of Immunology, PLA, Third Military Medical University, 30# Gaotanyan Street, District Shipingba, Chongqing 400038, PR China † Contributed equally Full list of author information is available at the end of the article BioMed Central © 2010 Li et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduc Li et al. Arthritis Research & Therapy 2010, 12:R81 http://arthritis-research.com/content/12/3/R81 Page 2 of 12 vival of CD4+ T cells has been shown to affect the behavior of synovial fibroblasts through the cell-to-cell contact and the secretion of proinflammatory factors such as Th1 and Th17 cytokines [9,10], ultimately contributing to the maintenance and exacerbation of inflammation in RA [11]. Although elevated levels of antiapoptotic proteins such as the Bcl-2 family have been found in these T cells [12], the possible mechanism underlying the impaired apoptosis of T cells in RA remains largely unclear. MicroRNAs (miRNAs) are about 22 nucleotide (nt) non-coding RNA that regulate mRNA expression at the posttranscriptional level for degradation or translational repression, which have been found to control cell division, differentiation and death [13]. To date, thousands of miRNAs have been identified in mammalian genomes, and up to 30% of human genes are regulated by them [14]. Recently, miRNAs have been recognized as a novel player in normal immune function and inflammation [15]. In particular, T cell-mediated immune responses are associated with changes in the expression of specific miRNAs. CD4+ T cells have also been found to express different miRNAs subsets that are linked to cell differentiation, maturation, activation and function [16-19]. Notably, a growing number of reports have revealed that deregulation of miRNA expression contributes to human autoimmune diseases including psoriasis and systemic lupus erythematosus [20,21], in which expression of a set of altered miRNAs are identified. In RA patients, increased expression of miR-146a, miR-155, miR-132 and miR-16 have been found in peripheral blood mononuclear cells (PBMCs) [22]. Recently, analysis of miRNA expression profile has revealed that miR-223 is overexpressed in peripheral T cells of RA patients [23]. Moreover, there is evidence that proliferation of fibroblast-like synoviocytes is regulated by miR-124a [24]. The abnormal expression of miR-146 and miR-155 is also found in the synovium of RA patients, while the expression of these miRNAs is markedly upregulated in SF after stimulation with TNF-α and IL-1β [25,26], which indicate that the inflammatory milieu may alter the miRNAs expression in joint tissue. Although a set of altered expression miRNAs have been identified in synovial tissue or PBMC of patients with RA, the functions of these dysregulated miRNAs remain largely unclear. In particular, both the expression profile and the roles of miRNAs in CD4+ T cells of RA patients have not been characterized. In this study, we revealed that miR-146a expression was upregulated in CD4+ T cells from RA patients, exhibiting a close correlation with increased levels of TNF-α. Furthermore, we demonstrated that the increased expression of miR-146a suppressed T cell apoptosis by regulating Fas-associated factor 1 (FAF1). These findings suggest that miR-146a in CD4+ T cells may play an important role in RA pathogenesis. Materials and methods Patients and control samples All patients in the study who first visited at Rheumatology Department of Southwest Hospital fulfilled the American College of Rheumatology criteria for the classification of RA. All patients in this study had been diagnosed without other complications. The patients had never been treated with oral or intra-articular corticosteroids, disease-modifying antirheumatic drugs (DMARDs) or other immunosuppressive drugs. However, nonsteroidal anti-inflammatory drugs and other symptomatic treatments were given to some patients. Samples from healthy donors were collected from 12 volunteers, half of which were men and half women. Serum and peripheral blood samples were collected from RA patients and healthy donors. SF from RA patients was collected by routine knee joint paracentesis. At least 10 ml of SF and 20 ml of peripheral blood anticoagulated by heparin were collected. All study protocols and consent forms were approved by the Institutional Medical Ethics Committee of Third Military Medical University. Written permission was obtained from all subjects who participated in the study. Microarray analysis CD4+ T cells were purified from the SF of two RA patients and peripheral blood of one healthy donor using microbeads (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). Total RNA of these CD4+ T cells was prepared using the mirVana miRNA Isolation Kit (Ambion, Austin, Texas, USA) for miRNA microarray analysis and realtime RT-PCR assay. Hybridization was carried out using a miRCURYTM Array microarray kit (Exqion, Vedbaek, Denmark), which contained 461 mature miRNA probes. The Affymetrix GeneChip (Human Genome U133 Plus 2.0 Array, Affymetrix, Santa Clara, CA) was used to analyze over 47,000 transcripts of Jurkat T cells before and after transfection with FUGW-FF3, FUGW-miR-146a or FUGW-miR-146a sponge. The expression data and gene annotations were stored in NCBI's Gene Expression Omnibus (GEO) database, accession no. [GEO:GSE21118] and [GEO:GSE21132] [27]. Quantitative RT-PCR analysis Quantitative RT-PCR assays were performed using a TaqMan® MicroRNA Assays kit (Applied Biosystems, Carlsbad, CA, USA) for the mature miRNA according to manufacturer's instructions. RUN6B small nuclear RNA was quantified as a control to normalize differences in total RNA levels. Specific primers to FAF1 for real-time Li et al. Arthritis Research & Therapy 2010, 12:R81 http://arthritis-research.com/content/12/3/R81 Page 3 of 12 PCR were designed using Roche's Applied Science Universal Probe Library Assay Design Center (Roche, Indianapolis, Indiana, USA) [28]; β-actin was quantified as the control. The primer sequences for FAF1 were as follows: 5'-CAGCGGGAGTACAACCTGA-3' (forward) and 5'AAGGTCATACACATTTCTCTTTACCTC-3' (reverse). The quantitative RT-PCR was performed by using Opticon-2 Detection System (Applied Biosystems, Carlsbad, CA, USA). All reactions were run in duplicate and repeated three times. A threshold cycle was determined in the exponential phases of amplification, and the comparative threshold cycle method was calculated with 2ΔΔT and used to calculate the relative gene expression. The value of each control sample was set at one and was used to calculate the fold change in target genes. Cloning of miR-146a gene and construction of vectors A 452 bp sequence containing the miR-146a hairpin (nucleotides 4721752-4722203 of chromosome5-