Takahashi A, de Andrés M C, Hashimoto K, Itoi E, Oreffo R O C
Bone and Joint Research Group, Centre for Human Development Stem Cells and Regeneration, Institute of Developmental Science, University of Southampton Medical School, Southampton, UK; Department of Orthopaedic Surgery, Tohoku University School of Medicine, Sendai, Japan.
Bone and Joint Research Group, Centre for Human Development Stem Cells and Regeneration, Institute of Developmental Science, University of Southampton Medical School, Southampton, UK.
Osteoarthritis Cartilage. 2015 Nov;23(11):1946-54. doi: 10.1016/j.joca.2015.02.168.
To determine whether altered IL8 methylation status is associated with increased expression of IL8 in human osteoarthritic (OA) chondrocytes.
IL8 expression levels and the percentage CpG methylation in human chondrocytes were quantified by qRT-PCR and pyrosequencing in OA patients and in non-OA osteoporotic controls. The effect of CpG methylation on IL8 promoter activity was determined using a CpG-free vector; co-transfections with expression vectors encoding nuclear factor-kappa B (NF-κB), AP-1 and C/EBP were subsequently undertaken to analyse for IL8 promoter activity in response to changes in methylation status.
IL8 expression in OA patients was 37-fold higher than in osteoporotic controls. Three CpG sites in the IL8 promoter were significantly demethylated in OA patients. Multiple regression analysis revealed that the degree of methylation of the CpG site located at -116-bp was the strongest predictor of IL8 expression. In vitro DNA methylation was noted to decrease IL8 promoter basal activity. Furthermore, NF-κB, AP-1 and C/EBP strongly enhanced IL8 promoter activity whilst DNA methylation inhibited the effects of these three transcription factors.
The present study demonstrates the key role of DNA methylation status on the expression of IL8 in human chondrocytes. We demonstrate a quantitative relationship between percentage methylation and gene expression within clinical samples. These studies provide direct evidence linking the activation of IL8, DNA demethylation and the induction of the OA process with important therapeutic implications therein for patients with this debilitating disease.
确定白细胞介素8（IL8）甲基化状态改变是否与人类骨关节炎（OA）软骨细胞中IL8表达增加有关。
通过定量逆转录聚合酶链反应（qRT-PCR）和焦磷酸测序对OA患者和非OA骨质疏松症对照者的人软骨细胞中IL8表达水平和CpG甲基化百分比进行定量。使用无CpG载体确定CpG甲基化对IL8启动子活性的影响；随后与编码核因子-κB（NF-κB）、激活蛋白-1（AP-1）和CCAAT增强子结合蛋白（C/EBP）的表达载体共转染，以分析IL8启动子活性对甲基化状态变化的反应。
OA患者中IL8表达比骨质疏松症对照者高37倍。OA患者中IL8启动子的三个CpG位点显著去甲基化。多元回归分析显示，位于-116-bp处的CpG位点甲基化程度是IL8表达的最强预测因子。体外DNA甲基化可降低IL8启动子基础活性。此外，NF-κB、AP-1和C/EBP强烈增强IL8启动子活性，而DNA甲基化抑制这三种转录因子的作用。
本研究证明了DNA甲基化状态对人软骨细胞中IL8表达的关键作用。我们证明了临床样本中甲基化百分比与基因表达之间的定量关系。这些研究提供了直接证据，将IL8激活、DNA去甲基化与OA进程的诱导联系起来，对这种使人衰弱的疾病患者具有重要的治疗意义。
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