Rezaei Ramazan, Mahmoudi Mahdi, Gharibdoost Farhad, Kavosi Hoda, Dashti Navid, Imeni Vahideh, Jamshidi Ahmadreza, Aslani Saeed, Mostafaei Shayan, Vodjgani Mohammad
Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran.
Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Int J Rheum Dis. 2017 Oct;20(10):1551-1561. doi: 10.1111/1756-185X.13175. Epub 2017 Sep 26.
The aim of the current study was to evaluate if methylation status of CpG sites of interferon regulatory factor 7 (IRF7) promoter in peripheral blood mononuclear cells (PBMCs) of systemic sclerosis (SSc) patients is involved in pathogenesis of the disease.
PBMCs were isolated from whole blood of 50 SSc patients and 30 controls. After the extraction of total RNA and DNA contents from PBMCs, complementary DNA (cDNA) was synthesized. Afterwards, quantitative analysis of IRF7 messenger RNA (mRNA) was conducted by real-time polymerase chain reaction (PCR). To evaluate the methylation status of the promoter region of IRF7 gene, PCR products of bisulfite-treated DNA from SSc patients and controls were sequenced.
The mRNA expression of IRF7 in PBMCs from patients compared with controls was significantly upregulated. While limited cutaneous SSc patients expressed the mRNA of IRF7 higher than controls, the diffuse cutaneous SSc group did not demonstrate significantly increased expression in comparison to controls. Insignificant promoter hypomethylation of IRF7 was observed in SSc patients compared with the control group. However, CpG2 hypomethylation was significantly associated with increased SSc risk. Furthermore, overall promoter methylation and mRNA level of IRF7 were significantly correlated with each other. Nonetheless, none of them correlated with Rodnan score of SSc patients. There was significant difference in IRF7 mRNA expression between CpG8 methylated and unmethylated SSc patients. Moreover, the difference of methylation and expression was not significant between anti-nuclear antibody (ANA)-positive and ANA-negative SSc patients.
It is suggested that hypomethylation of the IRF7 promoter might play a role in SSc pathogenesis, probably through promoting the IRF7 expression in PBMCs of patients with SSc.
本研究旨在评估系统性硬化症（SSc）患者外周血单个核细胞（PBMCs）中干扰素调节因子7（IRF7）启动子的CpG位点甲基化状态是否参与该疾病的发病机制。
从50例SSc患者和30例对照者的全血中分离PBMCs。从PBMCs中提取总RNA和DNA后，合成互补DNA（cDNA）。随后，通过实时聚合酶链反应（PCR）对IRF7信使核糖核酸（mRNA）进行定量分析。为评估IRF7基因启动子区域的甲基化状态，对SSc患者和对照者经亚硫酸氢盐处理的DNA的PCR产物进行测序。
与对照组相比，患者PBMCs中IRF7的mRNA表达显著上调。局限性皮肤型SSc患者的IRF7 mRNA表达高于对照组，而弥漫性皮肤型SSc组与对照组相比未显示出显著增加的表达。与对照组相比，SSc患者中观察到IRF7启动子低甲基化不显著。然而，CpG2低甲基化与SSc风险增加显著相关。此外，IRF7的总体启动子甲基化和mRNA水平彼此显著相关。尽管如此，它们均与SSc患者的罗德南评分无关。CpG8甲基化和未甲基化的SSc患者之间IRF7 mRNA表达存在显著差异。此外，抗核抗体（ANA）阳性和ANA阴性的SSc患者之间甲基化和表达的差异不显著。
提示IRF7启动子的低甲基化可能在SSc发病机制中起作用，可能是通过促进SSc患者PBMCs中IRF7的表达。
