Department of Internal Medicine and Arthritis and Autoimmunity Research Center, School of Medicine, Catholic University of Daegu, Daegu 705-718, Korea.
Mol Cells. 2013 Apr;35(4):298-304. doi: 10.1007/s10059-013-2302-0. Epub 2013 Feb 26.
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease of unknown origin, which exhibits a complex heterogeneity in its pathophysiological background, resulting in differential responses to a range of therapies and poor long-term prognosis. RA synovial fibroblasts (RASFs) are key player cells in RA pathogenesis. Identification of DNA methylation biomarkers is a field that provides potential for improving the process of diagnosis and prognosis of various human diseases. We utilized a genome-wide technique, methylated DNA isolation assay (MeDIA), in combination with a high resolution CpG microarray for discovery of novel hypermethylated genes in RASFs. Thirteen genes (APEX1, EBF3, EGR2, EN1, IRX1, IRX6, KIF12, LHX2, MIPOL1, SGTA, SIN3A, TOLLIP, and ZHX2) with three consecutive hypermethylated probes were isolated as candidate genes through two CpG microarrays. Pyrosequencing assay was performed to validate the methylation status of TGF-β signaling components, EBF3 and IRX1 genes in RASFs and osteoarthritis (OA) SFs. Hypermethylation at CpG sites in the EBF3 and IRX1 genes was observed with a high methylation index (MI) in RASFs (52.5% and 41.4%, respectively), while a lower MI was observ ed in OASFs and h ealthy SFs (13.2% for EBF3 and 4.3% for IRX1). In addition, RT-PCR analysis showed a remarkable decrease in their mRNA expression in the RA group, compared with the OA or healthy control, and their reduction levels correlated with MI. The current findings suggest that methylation-associated down-regulation of EBF3 and IRX1 genes may play an important role in a pathogenic effect of TGF-β on RASFs. However, further clinical validation with large numbers of patients is needed in order to confirm our findings.
类风湿关节炎（RA）是一种病因不明的慢性、系统性炎症性疾病，其病理生理背景表现出复杂的异质性，导致对一系列治疗方法的反应不同，长期预后不佳。RA 滑膜成纤维细胞（RASFs）是 RA 发病机制中的关键细胞。鉴定 DNA 甲基化生物标志物是一个有潜力改善各种人类疾病诊断和预后过程的领域。我们利用全基因组技术，即甲基化 DNA 分离测定法（MeDIA），结合高分辨率 CpG 微阵列，发现 RASFs 中新型超甲基化基因。通过两个 CpG 微阵列，分离出 13 个具有三个连续超甲基化探针的基因（APEX1、EBF3、EGR2、EN1、IRX1、IRX6、KIF12、LHX2、MIPOL1、SGTA、SIN3A、TOLLIP 和 ZHX2）作为候选基因。焦磷酸测序法用于验证 TGF-β 信号通路成分 EBF3 和 IRX1 基因在 RASFs 和骨关节炎（OA）SFs 中的甲基化状态。在 RASFs 中，EBF3 和 IRX1 基因的 CpG 位点发生高甲基化，甲基化指数（MI）分别为 52.5%和 41.4%，而在 OASFs 和健康 SFs 中观察到较低的 MI（EBF3 为 13.2%，IRX1 为 4.3%）。此外，与 OA 或健康对照组相比，RA 组的这些基因的 mRNA 表达水平显著降低，且其降低水平与 MI 相关。这些发现表明，EBF3 和 IRX1 基因的甲基化相关下调可能在 TGF-β 对 RASFs 的致病作用中发挥重要作用。然而，需要对大量患者进行进一步的临床验证，以确认我们的发现。
