Methylation status of HPV16 E2-binding sites classifies subtypes of HPV-associated oropharyngeal cancers
Methylation status of HPV16 E2-binding sites classifies subtypes of HPV-associated oropharyngeal cancers
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DOI：
10.1002/cncr.29315
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发表时间：
2015-06-15
期刊：
影响因子：
6.2
通讯作者：
Doeberitz, Magnus von Knebel
中科院分区：
文献类型：
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作者：
Reuschenbach, Miriam;Huebbers, Christian U.;Doeberitz, Magnus von Knebel
人乳头瘤病毒（HPV） E2蛋白是致癌基因E6/E7的转录抑制因子，E2功能的丧失被认为是致癌的关键步骤。HPV整合到宿主基因组可能会破坏E2基因。此外，HPV上游调控区E2结合位点（E2BSs）的CpG二核苷酸甲基化可能干扰E2对E6和E7的转录抑制。作者假设E2BS的CpG甲基化状态鉴定了与E2基因完整性和病毒整合相关的HPV16型（HPV16）相关的口咽鳞状细胞癌（OPSCC）亚型。方法对57例hpv16相关的OPSCC患者的上游调控区（包括e2bs1、2、3和4）中10个CpG二核苷酸的甲基化进行亚硫酸盐焦磷酸测序分析。通过基因扩增和实时定量逆转录-聚合酶链反应分析E2水平。采用整合特异性聚合酶链反应法测定病毒整合。结果在E2BS3和e2bs4处鉴定出三个不同甲基化的亚组：1)完全甲基化（>80%）与完整E2基因的整合HPV基因组存在相关；2)中等甲基化水平（20%-80%），主要是带有完整E2的外泌型HPV基因组；3)无甲基化(
BACKGROUNDThe human papillomavirus (HPV) E2 protein is a transcriptional repressor of the oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2-binding sites (E2BSs) in the HPV upstream regulatory region may interfere with transcriptional repression of E6 and E7 by E2. The authors hypothesized that the CpG methylation status of E2BS identifies subtypes of HPV type 16 (HPV16)-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration.METHODSMethylation of 10 CpG dinucleotides within the upstream regulatory region, encompassing E2BSs 1, 2, 3, and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC cases. E2 status was analyzed by gene amplification and quantitative real-time reverse transcriptase-polymerase chain reaction. Viral integration was determined by integration-specific polymerase chain reaction methods.RESULTSThree subgroups with differential methylation at E2BS3 and E2BS 4 were identified: 1) complete methylation (>80%) associated with the presence of integrated HPV genomes with an intact E2 gene; 2) intermediate methylation levels (20%-80%) with predominantly episomal HPV genomes with intact E2; and 3) no methylation (