ABSTRACT

To evaluate the possible involvement of epigenetic modulation by HPV16-p16^INK4a in oral potentially malignant disorder (OPMD). We generated DNA-methylation profiles, according to p16^INK4a expression and HPV16 genotype (positive or negative), of OPMD samples and p16^INK4a-HPV16 negative samples (used as control), using reduced-representation bisulphite sequencing (RRBS-Seq- Illumina) technology. Twelve samples, four for each group, as follows: 1) p16^INK4a+ HPV16+; 2) p16^INK4a+ HPV16-; 3) p16^INK4a- HPV16-, were analysed in triplicate for DNA-methylation profiles. Fifty-four per cent of DMRs were hypermethylated and 46% were hypomethylated. An increase in methylation of loci in OPMD was independent of the presence of HPV. The hypermethylated genes in HPV+ samples were associated with signalling pathways such as NICD traffics to nucleus, signalling by NOTCH1 (p = 0.008), Interferon-gamma (p = 0.008) and Interleukin-6 signalling (p = 0.027). The hypomethylated genes in HPV infection were associated with TRAF3-dependent IRF activation pathway (p = 0.002), RIG-I/MDA5 mediated induction of IFN-alpha/beta pathways (p = 0.005), TRAF6 mediated IRF7 activation (p = 0.009), TRIF-mediated TLR3/TLR4 signalling (p = 0.011) and MyD88-independent cascade release of apoptotic factors (p = 0.011). Protein association analysis of DMRs in OPMD revealed 19 genes involved in the cell cycle regulation, immune system, and focal adhesion. Aberrantly methylated loci in OPMD were observed in p16^INK4a positive samples which suggests that a shift in global methylation status may be important for cancer progression. The results suggest that HPV infection in OPMD induces modulation of genes related to the immune system and regulation of the cellular cycle.

摘要

评估 HPV16-p16 ^INK4a表观遗传调控在口腔潜在恶性疾病 (OPMD) 中的可能作用。根据 p16 ^INK4a表达和 HPV16 基因型（阳性或阴性），我们使用减少代表性亚硫酸氢盐测序 (RRBS-Seq-Illumina) 生成了 OPMD 样本和 p16 ^INK4a -HPV16 阴性样本（用作对照）的DNA 甲基化谱技术。十二个样本，每组四个，如下： 1) p16 ^INK4a + HPV16+；2) p16 ^INK4a + HPV16-；3) p16 ^INK4a- HPV16-，一式三份分析DNA甲基化谱。54% 的 DMR 是高甲基化的，46% 是低甲基化的。OPMD 中基因座甲基化的增加与 HPV 的存在无关。HPV+ 样本中的高甲基化基因与信号通路相关，例如 NICD 向细胞核的交通、NOTCH1 (p = 0.008)、干扰素-γ (p = 0.008) 和白细胞介素-6 信号传导 (p = 0.027) 的信号传导。HPV 感染中的低甲基化基因与 TRAF3 依赖性 IRF 激活途径相关（p = 0.002）、RIG-I/MDA5 介导的 IFN-α/β 途径诱导（p = 0.005）、TRAF6 介导的 IRF7 激活（p = 0.009） , TRIF 介导的 TLR3/TLR4 信号 (p = 0.011) 和 MyD88 独立的凋亡因子级联释放 (p = 0.011)。OPMD 中 DMR 的蛋白质关联分析揭示了 19 个参与细胞周期调节、免疫系统和粘着斑的基因。在 p16 中观察到 OPMD 中的异常甲基化基因座^INK4a阳性样本表明全球甲基化状态的转变可能对癌症进展很重要。结果表明，OPMD中的HPV感染诱导了与免疫系统相关的基因的调节和细胞周期的调节。