Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis
Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis
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DOI：
10.1038/aps.2011.98
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发表时间：
2011-11-01
影响因子：
8.2
通讯作者：
Zhang, Jing-ge
中科院分区：
文献类型：
--
作者：
Fu, Li-hong;Ma, Chun-ling;Zhang, Jing-ge
目的：人白细胞介素-10（IL-10）是一种抑制炎症和调节免疫反应的关键细胞因子，其启动子含有一个具有CpG基序的种间保守序列。本研究旨在探讨类风湿关节炎（RA）患者IL 10基因CpG基序甲基化对IL 10表达的调控作用。收集20名RA患者和20名健康对照的外周血单核细胞（PBMCs）。6例患者的PBMC在5-氮杂胞苷（5 μ mol/L）存在或不存在的情况下培养。采用RT-PCR和ELISA方法检测IL 10的mRNA和蛋白水平。通过焦磷酸测序确定IL 10启动子中CpG的甲基化。结果：在IL 10启动子区发现一个种间保守序列，该序列在IL 10基因启动子区的转录水平上具有明显的种间保守性。在RA患者和健康对照的PBMC中，上游CpG在-408、-387、-385和-355 bp处高甲基化。相反，RA患者中-145处的近端CpG的低甲基化程度比健康对照高得多（P=0.016），这与较高的IL 10 mRNA和血清水平相关。在5-氮胞苷处理的PBMC中，CpG基序被去甲基化，IL 10 mRNA和蛋白的表达水平显著增加。结论：IL 10启动子近端CpG甲基化可能参与RA基因转录调控。
Aim: The promoter of human interleukin-10 (IL10), a cytokine crucial for suppressing inflammation and regulating immune responses, contains an interspecies-conserved sequence with CpG motifs. The aim of this study was to investigate whether methylation of CpG motifs could regulate the expression of IL10 in rheumatoid arthritis (RA).Methods: Bioinformatic analysis was conducted to identify the interspecies-conserved sequence in human, macaque and mouse IL10 genes. Peripheral blood mononuclear cells (PBMCs) from 20 RA patients and 20 health controls were collected. The PBMCs from 6 patients were cultured in the presence or absence of 5-azacytidine (5 mu mol/L). The mRNA and protein levels of IL10 were examined using RT-PCR and ELISA, respectively. The methylation of CpGs in the IL10 promoter was determined by pyrosequencing. Chromatin immunoprecipitation (ChIP) assays were performed to detect the cyclic AMP response element-binding protein (CREB)-DNA interactions.Results: One interspecies-conserved sequence was found within the IL10 promoter. The upstream CpGs at -408, -387, -385, and -355 bp were hypermethylated in PBMCs from both the RA patients and healthy controls. In contrast, the proximal CpG at -145 was hypomethylated to much more extent in the RA patients than in the healthy controls (P=0.016), which was correlated with higher IL10 mRNA and serum levels. In the 5-azacytidine-treated PBMCs, the CpG motifs were demethylated, and the expression levels of IL10 mRNA and protein was significantly increased. CHIP assays revealed increased phospho-CREB binding to the IL10 promoter.Conclusion: The methylation of the proximal CpGs in the IL10 promoter may regulate gene transcription in RA.