人乳头瘤病毒16型长控制区内CpG位点甲基化状态对人乳头瘤病毒16型阳性头颈癌细胞的影响
Effects of Methylation Status of CpG Sites within the HPV16 Long Control Region on HPV16-Positive Head and Neck Cancer Cells.
作者信息
Zhang Chunlin, Deng Zeyi, Pan Xiaoli, Uehara Takayuki, Suzuki Mikio, Xie Minqiang
机构信息
Department of Otorhinolaryngology, Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China; Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, Okinawa, Japan; Department of Otorhinolaryngology, Head and Neck Surgery, Affiliated hospital of Zunyi Medical University, Zunyi, China.
Department of Otorhinolaryngology, Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China; Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, Okinawa, Japan.
出版信息
PLoS One. 2015 Oct 28;10(10):e0141245. doi: 10.1371/journal.pone.0141245. eCollection 2015.
OBJECTIVE
To map comprehensively the methylation status of the CpG sites within the HPV16 long control region (LCR) in HPV-positive cancer cells, and to explore further the effects of methylation status of HPV16 LCR on cell bioactivity and E6 and E7 expression. In addition, to analyze the methylation status of the LCR in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) patients.
METHODS AND MATERIALS
Methylation patterns of HPV16 LCR in UM-SCC47, CaSki, and SiHa cells and HPV16-positiive OPSCC specimens were detected by bisulfite-sequencing PCR and TA cloning. For cells treated with 5-aza-2'-deoxycytidine and E6 and E7 knockdown, MTS and trypan blue staining, annexin-V and 7-AAD staining, and prodidium iodide were used to evaluate cell growth and cell proliferation, cell apoptosis, and cell cycle arrest, respectively. E6 and E7 mRNA and protein expression were analyzed by quantitative real-time PCR and immunocytochemistry, respectively.
RESULTS
Hypermethylation status of the LCR in UM-SCC47 (79.8%) and CaSki cells (90.0%) and unmethylation status of the LCR in SiHa cells (0%) were observed. Upon demethylation, the cells with different methylation levels responded differently during growth, apoptosis, and cell cycle arrest, as well as in terms of their E6 and E7 expression. In HPV16-positive OPSCC patients, the methylation rates were 9.5% in the entire LCR region, 13.9% in the 5'-LCR, 6.0% in the E6 enhancer, and 9.5% in the p97 promoter, and hypermethylation of p97 promoter was found in a subset of cases (20.0%, 2/10).
CONCLUSIONS
Our study revealed two different methylation levels of the LCR in HPV16-positive cancer cells and OPSCC patients, which may represent different carcinogenesis mechanisms of HPV-positive cancers cells. Demethylating the meCpGs in HPV16 LCR might be a potential target for a subgroup of HPV16-positive patients with head and neck squamous cell carcinoma.
目的
全面绘制人乳头瘤病毒16型（HPV16）阳性癌细胞中HPV16长控制区（LCR）内CpG位点的甲基化状态，并进一步探讨HPV16 LCR甲基化状态对细胞生物活性以及E6和E7表达的影响。此外，分析HPV阳性口咽鳞状细胞癌（OPSCC）患者中LCR的甲基化状态。
方法和材料
通过亚硫酸氢盐测序PCR和TA克隆检测UM-SCC47、CaSki和SiHa细胞以及HPV16阳性OPSCC标本中HPV16 LCR的甲基化模式。对于用5-氮杂-2'-脱氧胞苷处理以及E6和E7基因敲低的细胞，分别使用MTS和台盼蓝染色、膜联蛋白-V和7-氨基放线菌素D染色以及碘化丙啶来评估细胞生长和细胞增殖、细胞凋亡以及细胞周期阻滞。分别通过定量实时PCR和免疫细胞化学分析E6和E7 mRNA及蛋白表达。
结果
观察到UM-SCC47细胞（79.8%）和CaSki细胞（90.0%）中LCR的高甲基化状态以及SiHa细胞中LCR的未甲基化状态（0%）。去甲基化后，不同甲基化水平的细胞在生长、凋亡、细胞周期阻滞以及E6和E7表达方面表现出不同的反应。在HPV16阳性OPSCC患者中，整个LCR区域的甲基化率为9.5%，5'-LCR为13.9%，E6增强子为6.0%，p97启动子为9.5%，并且在一部分病例（20.0%，2/10）中发现p97启动子存在高甲基化。
结论
我们的研究揭示了HPV16阳性癌细胞和OPSCC患者中LCR存在两种不同的甲基化水平，这可能代表HPV阳性癌细胞不同的致癌机制。对HPV16 LCR中的甲基化CpG进行去甲基化可能是一部分HPV16阳性头颈部鳞状细胞癌患者的潜在治疗靶点。
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