Identification of novel markers in rheumatoid arthritis through integrated analysis of DNA methylation and microRNA expression
Highlights
► We have identified novel genes with altered DNA methylation in RASF. ► We have identified novel miRNAs with altered expression levels genes in RASF. ► We identified different relationships between methylation, miRNA and expression data. ► We developed a novel method to integrate the complexity of gene dysregulation in RA.
Introduction
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by the progressive destruction of the joints. RA pathogenesis involves a variety of cell types, including several lymphocyte subsets, dendritic cells, osteoclasts and synovial fibroblasts (SFs). In healthy individuals, SFs are essential to keep the joints in shape, doing so by providing nutrients, facilitating matrix remodeling and contributing to tissue repair [1]. In contrast to normal SFs or those isolated from patients with osteoarthritis (osteoarthritis synovial fibroblasts, OASFs), rheumatoid arthritis synovial fibroblasts (RASFs) show activities associated with an aggressive phenotype, like upregulated expression of protooncogenes, specific matrix-degrading enzymes, adhesion molecules, and cytokines [2]. Differences in phenotype and gene expression between RASFs and their normal counterparts reflect a profound change in processes involved in gene regulation at the transcriptional and post-transcriptional levels. The first group comprises epigenetic mechanisms, like DNA methylation, whilst miRNA control constitutes one of the best studied mechanisms of the second.
DNA methylation takes place in cytosine bases followed by guanines. In relation with transcription, the repressive role of methylation at CpG sites located at or near the transcription start sites of genes, especially when those CpGs are clustered as CpG islands, is well established [3]. Methylation of CpGs located in other regions like gene bodies is also involved in gene regulation [4], [5]. At the other side of gene regulation lie microRNAs (miRNAs), a class of endogenous, small, non-coding regulatory RNA molecules that modulate the expression of multiple target genes at the post-transcriptional level and that are implicated in a wide variety of cellular processes and disease pathogenesis [6].
The study of epigenetic- and miRNA-mediated alterations in association with disease is becoming increasingly important as these processes directly participate in the generation of aberrant profiles of gene expression ultimately determining cell function and are pharmacologically reversible. Epigenetics is particularly relevant in autoimmune rheumatic diseases as it is highly dependent on environmental effects. As indicated above, both genetics and environmental factors contribute to ethiopathology of autoimmune rheumatic disorders. This double contribution is typically exemplified by the partial concordance in monozygotic twins (MZ) [7], [8]. It is of inherent interest to identify autoimmune disease phenotypes for which the environment plays a critical role [9]. Many environmental factors, including exposure to chemicals, tobacco smoke, radiation, ultraviolet (UV) light and infectious agents among other external factors, are associated with the development of autoimmune rheumatic disorders [10]. Most of these environmental factors are now known to directly or indirectly induce epigenetic changes, which modulate gene expression and therefore associate with changes in cell function. For this reason, epigenetics provides a source of molecular mechanisms that can explain the environmental effects on the development of autoimmune disorders [11]. The close relationship between environment and epigenetic status and autoimmune rheumatic disease is also exemplified by using animal models [12]. This type of studies is also essential for the identification of novel clinical markers for disease onset, progression and response to treatments.
In this line, initial reports demonstrated hypomethylation-associated reactivation of endogenous retroviral element L1 in the RA synovial lining at joints [13]. Additional sequences have since been found to undergo hypomethylation in RASFs, like IL-6 [14] and CXCL12 [15]. Candidate gene analysis has also enabled genes to be identified that are hypermethylated in RASFs [16]. More recently, DNA methylation profiling of RASFs versus OASFs has led to the identification of a number of hypomethylated and hypermethylated genes [17]. With respect to miRNAs, reduced miR-34a levels have been linked with increased resistance of RASFs to apoptosis [18], and lower miR-124a levels in RASFs impact its targets, CDK-2 and MCP-1 [19]. Conversely, miR-203 shows increased expression in RASFs [20]. Interestingly, overexpression of this miR-203 is demethylation-dependent, highlighting the importance of investigating multiple levels of regulation and the need to use integrated strategies that consider interconnected mechanisms.
In this study, we have performed the first integrated comparison of DNA methylation and miRNA expression data, together with mRNA expression data from RASFs versus OASFs (Fig. 1) in order to investigate the relevance of these changes in these cells and to overcome the limitations of using a small number of samples. Our analysis identifies novel targets of DNA methylation- and miRNA-associated dysregulation in RA. Integration of the analysis of these two datasets suggests the existence of several genes for which the two mechanisms could act in the same or in opposite directions.
Section snippets
Subjects and sample preparation
Fibroblast-like synoviocytes (FLSs) were isolated from synovial tissues extracted from RA and OA patients at the time of joint replacement in the Department of Rheumatology of Leiden University Medical Center. All RA patients met the 1987 criteria of the American College of Rheumatology. Before tissue collection, permission consistent with the protocol of the Helsinki International Conference on Harmonisation Good Clinical Practice was obtained. All individuals gave informed consent. Synovial
Comparison of DNA methylation patterns between RASF and OASF reveals both hypomethylation and hypermethylation of key genes
We performed high-throughput DNA methylation screening to compare SF samples from six RA and six OA patients. To this end, we used a methylation bead array that allows the interrogation of >450,000 CpG sites across the entire genome covering 99% of RefSeq genes. Statistical analysis of the combined data from the 12 samples showed that 2571 CpG sites, associated with 1240 different genes, had significant differences in DNA methylation between RASFs and OASFs (median β differences > 0.10, p
Discussion
In this study we have identified novel dysregulated targets in rheumatoid arthritis (RA) synovial fibroblasts at the DNA methylation and miRNA expression levels. By using a double approach and integrated analysis of the DNA methylation, miRNA expression and mRNA expression data we have established a new pipeline for investigating the complexity of gene dysregulation in the context of this disease when using primary samples. As indicated above, dysregulation of gene expression arises from a
Acknowledgments
We would like to thank Dr. Gary Firestein for sharing the raw data of his DNA methylation study with us. We would also like to thank José Luis Pablos for his valuable feedback on his expression dataset. This work was supported by grant SAF2011-29635 from the Spanish Ministry of Science and Innovation, grant from Fundación Ramón Areces and grant 2009SGR184 from AGAUR (Catalan Government). LR is supported by a PFIS predoctoral fellowship and AI was supported by a AGAUR predoctoral fellowship.
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