Department of pharmacy, Luohe Medical College, 148 Daxue-Road, Luohe 462002, China.
Cell Oncol (Dordr). 2013 Jul;36(4):323-31. doi: 10.1007/s13402-013-0137-5. Epub 2013 Jun 6.
Previous studies have shown a down-regulation of the gene encoding cyclin-dependent kinase 10 (CDK10) in hepatocellular carcinomas. Here we provide evidence that down-regulation of the CDK10 gene is mediated by promoter hypermethylation in primary human nasopharyngeal carcinomas (NPC) and NPC-derived cell lines.
RT-PCR, Western blotting, methylation-specific PCR and bisulfite sequencing were performed to assess the expression and methylation status of the CDK10 gene in primary NPC samples, NPC-derived cell lines and patient-derived peripheral blood samples. The NPC-derived cell line CNE-2 was selected for treatment with a methylation inhibitor to restore CDK10 expression. In addition, cell proliferation, invasion and colony formation assays were performed to assess the inhibitory effects of ectopic CDK10 expression in CNE-2 cells.
Down-regulation of CDK10 expression in primary NPC samples (23/40, 57.5%) was found to be significantly correlated with the methylation status of its promoter CpG island (21/40, 52.5%). Demethylation by 5-aza-dC treatment led to reactivation of the CDK10 gene in the CNE-2 cell line. Additionally, exogenous expression of CDK10 in CNE-2 cells strongly suppressed its growth, invasion and colony formation capacities. The high sensitivity (15/40, 37.5%) and specificity (0% false positives) of detecting CDK10 promoter hypermethylation in NPC patient-derived peripheral blood samples suggest that it could be employed as an epigenetic marker for noninvasive cancer diagnosis and recurrence screening.
Our findings implicate that aberrant methylation of the CDK10 gene promoter occurs frequently in NPC, and that reactivation of CDK10 might be utilized as a novel epigenetic strategy for the treatment of NPC patients.
先前的研究表明，细胞周期蛋白依赖性激酶 10（CDK10）的基因在肝细胞癌中下调。在这里，我们提供的证据表明，CDK10 基因的下调是由原发性人鼻咽癌（NPC）和 NPC 衍生细胞系中的启动子超甲基化介导的。
通过 RT-PCR、Western blot、甲基化特异性 PCR 和亚硫酸氢盐测序，评估 CDK10 基因在原发性 NPC 样本、NPC 衍生细胞系和患者衍生外周血样本中的表达和甲基化状态。选择 NPC 衍生细胞系 CNE-2 进行甲基化抑制剂处理以恢复 CDK10 表达。此外，进行细胞增殖、侵袭和集落形成测定以评估 CNE-2 细胞中外源 CDK10 表达的抑制作用。
在原发性 NPC 样本中发现 CDK10 表达下调（23/40，57.5%）与启动子 CpG 岛的甲基化状态显著相关（21/40，52.5%）。5-aza-dC 处理的去甲基化导致 CNE-2 细胞系中 CDK10 基因的重新激活。此外，CNE-2 细胞中外源 CDK10 的表达强烈抑制其生长、侵袭和集落形成能力。在 NPC 患者衍生外周血样本中检测 CDK10 启动子超甲基化的高灵敏度（15/40，37.5%）和特异性（0%假阳性）表明，它可作为非侵入性癌症诊断和复发筛查的表观遗传标记。
我们的研究结果表明，CDK10 基因启动子的异常甲基化在 NPC 中经常发生，并且重新激活 CDK10 可能被用作治疗 NPC 患者的新型表观遗传策略。
