Peng Hong, Chen Yuxiang, Gong Pinggui, Cai Longmei, Lyu Xiaoming, Jiang Qiang, Wang Jianguo, Lu Juan, Yao Kaitai, Liu Kunping, Li Jinbang, Li Xin
Department of Otorhinolaryngology at Nanfang Hospital, Cancer Research Institute, Southern Medical University, Guangzhou 510515, China.
Department of Otolaryngology-Head and Neck Surgery, The Second People's Hospital of Guangdong Province, Southern Medical University, Guangzhou 510515, China.
Oncotarget. 2016 Jun 28;7(26):40025-40037. doi: 10.18632/oncotarget.9474.
Phosphatase and tensin homolog (PTEN) is a major tumor suppressor and usually silenced via the deletion, insertion and mutation. We previously discovered its inactivation via aberrant CpG island methylation. Here, we provide further evidence that EBV latent membrane protein 1(LMP1) can induce a higher intensity of DNA methylation at PTEN CpG islands, inactivating PTEN at the cellular and molecular level. Initially, increased methylation intensity of PTEN CpG islands was observed in EBV-infected nasopharyngeal carcinoma (NPC) cells, accompanied by decreased PTEN expression. In NPC tissue samples showing the methylation at PTEN promoter, LMP1 was highly expressed in higher methylation intensity group relative to lower intensity group, and DNA methyltransferase 3b (DNMT3b) expression was positively correlated with LMP1 expression. Moreover, transfection of LMP1 gene into EBV-negative NPC cells demonstrated that LMP1 up-regulated DNMT3b expression, leading to a higher intensity of PTEN CpG island methylation. Mechanistically, computational prediction and luciferase reporter assay identified a functional NF-κB binding site on DNMT3b promoter and the mutated NF-κB binding site abolished LMP1-mediated DNMT3b activation. Chromatin immunoprecipitation displayed that NF-κB p65 subunit constitutively bound to DNMT3b promoter, supporting the activation of DNMT3b by EBV LMP1 via NF-κB signaling. Furthermore, the expression level of DNMT3b was observed to be increased in the nuclei of LMP1-expressing NPC cells, and a NF-κB inhibitor, PDTC, counteracted LMP1-mediated DNMT3b overexpression. Thus, this study first reports that LMP1-mediated NF-κB can up-regulate DNMT3b transcription, thereby leading to relatively higher methylation intensity at PTEN CpG islands, and ultimately silencing major tumor suppressor PTEN.
磷酸酶和张力蛋白同源物（PTEN）是一种主要的肿瘤抑制因子，通常通过缺失、插入和突变而沉默。我们之前发现其可通过异常的CpG岛甲基化而失活。在此，我们提供进一步的证据表明，EB病毒潜伏膜蛋白1（LMP1）可诱导PTEN CpG岛处更高强度的DNA甲基化，在细胞和分子水平使PTEN失活。最初，在EB病毒感染的鼻咽癌（NPC）细胞中观察到PTEN CpG岛甲基化强度增加，同时PTEN表达降低。在显示PTEN启动子甲基化的NPC组织样本中，相对于低甲基化强度组，LMP1在高甲基化强度组中高表达，且DNA甲基转移酶3b（DNMT3b）表达与LMP1表达呈正相关。此外，将LMP1基因转染到EB病毒阴性的NPC细胞中表明，LMP1上调DNMT3b表达，导致PTEN CpG岛甲基化强度更高。机制上，计算预测和荧光素酶报告基因检测在DNMT3b启动子上鉴定出一个功能性NF-κB结合位点，而突变的NF-κB结合位点消除了LMP1介导的DNMT3b激活。染色质免疫沉淀显示NF-κB p65亚基持续结合到DNMT3b启动子上，支持EB病毒LMP1通过NF-κB信号通路激活DNMT3b。此外，在表达LMP1的NPC细胞核中观察到DNMT3b表达水平升高，并且一种NF-κB抑制剂PDTC可抵消LMP1介导的DNMT3b过表达。因此，本研究首次报道LMP1介导的NF-κB可上调DNMT3b转录，从而导致PTEN CpG岛处甲基化强度相对更高，最终使主要肿瘤抑制因子PTEN沉默。
