Effects of Methylation Status of CpG Sites within the HPV16 Long Control Region on HPV16-Positive Head and Neck Cancer Cells.
Effects of Methylation Status of CpG Sites within the HPV16 Long Control Region on HPV16-Positive Head and Neck Cancer Cells.
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HPV16 长控制区内 CpG 位点甲基化状态对 HPV16 阳性头颈癌细胞的影响
DOI：
10.1371/journal.pone.0141245
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Xie M
中科院分区：
文献类型：
--
作者：
Zhang C;Deng Z;Pan X;Uehara T;Suzuki M;Xie M
目的全面绘制hpv阳性癌细胞HPV16长控制区（LCR） CpG位点的甲基化状态，进一步探讨HPV16长控制区甲基化状态对细胞生物活性和E6、E7表达的影响。此外，分析hpv阳性口咽鳞状细胞癌（OPSCC）患者LCR的甲基化状态。方法与材料采用亚硫酸盐测序PCR和TA克隆技术检测UM-SCC47、CaSki、SiHa细胞和HPV16阳性OPSCC标本中HPV16 LCR的甲基化模式。对5-aza-2′-脱氧胞苷和E6、E7敲低处理的细胞，分别用MTS和台锥蓝染色、annexin-V和7-AAD染色、碘化钠评价细胞生长和细胞增殖、细胞凋亡和细胞周期阻滞。采用实时荧光定量PCR和免疫细胞化学分别分析E6和E7 mRNA和蛋白的表达情况。结果观察到UM-SCC47细胞和CaSki细胞LCR的高甲基化状态（79.8%）和SiHa细胞LCR的非甲基化状态（0%）。在去甲基化后，不同甲基化水平的细胞在生长、凋亡、细胞周期停滞以及E6和E7的表达方面有不同的反应。在hpv16阳性的OPSCC患者中，整个LCR区域的甲基化率为9.5%,5 ' -LCR区域为13.9%，E6增强子为6.0%，p97启动子为9.5%，在一部分病例中发现p97启动子超甲基化（20.0%,2/10）。结论hpv16阳性癌细胞和OPSCC患者中LCR的两种不同甲基化水平，可能代表了hpv阳性癌细胞的不同癌变机制。HPV16 LCR中mecpg的去甲基化可能是HPV16阳性头颈部鳞状细胞癌患者亚组的潜在靶标。
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.