University of Southampton Medical School, Southampton, UK, and Tohoku University Graduate School of Medicine, Sendai, Japan.
Arthritis Rheumatol. 2014 Nov;66(11):3040-51. doi: 10.1002/art.38774.
To investigate whether the changes in collagen gene expression in osteoarthritic (OA) human chondrocytes are associated with changes in the DNA methylation status in the COL2A1 enhancer and COL9A1 promoter.
Expression levels were determined using quantitative reverse transcription-polymerase chain reaction, and the percentage of DNA methylation was quantified by pyrosequencing. The effect of CpG methylation on COL9A1 promoter activity was determined using a CpG-free vector; cotransfections with expression vectors encoding SOX9, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α were carried out to analyze COL9A1 promoter activities in response to changes in the methylation status. Chromatin immunoprecipitation assays were carried out to validate SOX9 binding to the COL9A1 promoter and the influence of DNA methylation.
Although COL2A1 messenger RNA (mRNA) levels in OA chondrocytes were 19-fold higher than those in the controls, all of the CpG sites in the COL2A1 enhancer were totally demethylated in both samples. The levels of COL9A1 mRNA in OA chondrocytes were 6,000-fold lower than those in controls; 6 CpG sites of the COL9A1 promoter were significantly hypermethylated in OA patients as compared with controls. Treatment with 5-azadeoxycitidine enhanced COL9A1 gene expression and prevented culture-induced hypermethylation. In vitro methylation decreased COL9A1 promoter activity. Mutations in the 5 CpG sites proximal to the transcription start site decreased COL9A1 promoter activity. Cotransfection with SOX9 enhanced COL9A1 promoter activity; CpG methylation attenuated SOX9 binding to the COL9A1 promoter.
This first demonstration that hypermethylation is associated with down-regulation of COL9A1 expression in OA cartilage highlights the pivotal role of epigenetics in OA, involving not only hypomethylation, but also hypermethylation, with important therapeutic implications for OA treatment.
研究骨关节炎（OA）患者软骨细胞中胶原蛋白基因表达的变化是否与 COL2A1 增强子和 COL9A1 启动子中的 DNA 甲基化状态变化有关。
采用定量逆转录聚合酶链反应测定表达水平，采用焦磷酸测序法定量测定 DNA 甲基化百分比。通过无 CpG 载体确定 CpG 甲基化对 COL9A1 启动子活性的影响；转染 Sox9、缺氧诱导因子 1α（HIF-1α）和 HIF-2α表达载体，分析 COL9A1 启动子活性对甲基化状态变化的反应。进行染色质免疫沉淀试验以验证 Sox9 与 COL9A1 启动子的结合以及 DNA 甲基化的影响。
OA 软骨细胞中 COL2A1 信使 RNA（mRNA）水平是对照组的 19 倍，但 COL2A1 增强子的所有 CpG 位点在两个样本中均完全去甲基化。OA 软骨细胞中 COL9A1 mRNA 水平比对照组低 6000 倍；与对照组相比，OA 患者 COL9A1 启动子的 6 个 CpG 位点显著超甲基化。5-氮杂脱氧胞苷处理可增强 COL9A1 基因表达并防止培养诱导的过度甲基化。体外甲基化降低了 COL9A1 启动子活性。靠近转录起始位点的 5 个 CpG 位点的突变降低了 COL9A1 启动子活性。共转染 Sox9 增强了 COL9A1 启动子活性；CpG 甲基化减弱了 Sox9 与 COL9A1 启动子的结合。
本研究首次证明，OA 软骨中 COL9A1 表达下调与过度甲基化有关，这突显了表观遗传学在 OA 中的关键作用，不仅涉及低甲基化，还涉及过度甲基化，对 OA 治疗具有重要的治疗意义。
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