背景：口腔鳞状细胞癌（OSCC）是全世界最常见的癌症类型之一。尽管之前已经观察到 5-甲基胞嘧啶 (5mC) 和 5-羟甲基胞嘧啶 (5hmC) 的整体丢失，但 OSCC 中 5mC 和 5hmC 的全基因组、单碱基分辨率和同步作图仍未实现。同样，5mC 和 5hmC 如何共同导致 OSCC 基因表达异常的机制在很大程度上尚未被探索。使用并行全基因组亚硫酸氢盐测序 (WGBS) 和全基因组氧化亚硫酸氢盐测序 (oxWGBS)，我们在配对的原代 OSCC 样本及其正常邻近组织 (NAT) 中以单核苷酸分辨率表征了 5mC 和 5hmC 谱。我们还使用多组学分析分析了 5mC 和 5hmC 修饰对 OSCC 差异基因表达的影响。结果：在 OSCC 样本中观察到各个基因组区域的 5mC 和 5hmC 总体降低。在 OSCC 启动子区域，共鉴定出 6,921 个差异甲基化区域和 1,024 个差异羟甲基化区域。有趣的是，与5mC和5hmC的双向修饰相比，启动子处5mC和5hmC的单向修饰与基因表达的更大变化相关。此外，启动子处带有 5mC 和 5hmC 单向修饰的基因富含信号通路，如细胞增殖、细胞分化和受体酪氨酸激酶通路，这些通路对于肿瘤发生至关重要。最后，带有 5mC 和 5hmC 启动子单向修饰的前 20 个基因的分组表达特征往往与头颈鳞状细胞癌某些亚型的临床结果相关。结论：使用平行 WGBS 和 oxWGBS 分析，我们观察到 OSCC 中各个基因组区域的 5mC 和 5hmC 修饰总体减少。启动子处 5mC 和 5hmC 的单向修饰与 OSCC 组织中基因表达的增强变化相关。此外，这种在启动子处带有 5mC 和 5hmC 单向修饰的差异表达基因可能与 OSCC 的结果具有临床相关性。版权所有 © 2023 赵、朱、龚、马、熊、苏、万和王。

Background: Oral squamous cell carcinoma (OSCC) is one of the most common types of cancer worldwide. Although overall losses of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been previously observed, a genome-wide, single-base-resolution, and simultaneous mapping of 5mC and 5hmC in OSCC is still unaccomplished. Similarly, the mechanism of how 5mC and 5hmC collectively lead to abnormal gene expression in OSCC is largely unexplored. Using parallel whole-genome bisulfite sequencing (WGBS) and whole-genome oxidative bisulfite sequencing (oxWGBS), we characterized 5mC- and 5hmC-profiles at single-nucleotide resolution in paired primary OSCC samples and their normal adjacent tissues (NATs). We also analyzed the effect of 5mC- and 5hmC-modifications on differential gene expression in OSCC using multi-omics analysis. Results: An overall reduction of both 5mC and 5hmC in various genomic regions have been observed in OSCC samples. At promoter regions, a total of 6,921 differentially methylated regions and 1,024 differentially hydroxymethylated regions were identified in OSCC. Interestingly, compared to bidirectional modification with 5mC and 5hmC, unidirectional modification with 5mC and 5hmC at the promoters is associated with bigger change in the gene expression. Additionally, genes bearing unidirectional modification with 5mC and 5hmC at the promoters are enriched in signaling pathways like cell proliferation, cell differentiation, and receptor tyrosine kinase pathway that are essential for the tumorigenesis. Finally, the grouped expression signature of top 20 genes bearing promoter-unidirectional-modification with 5mC and 5hmC tends to correlate with the clinical outcome of certain subtypes of head and neck squamous cell carcinoma. Conclusion: Using parallel WGBS and oxWGBS analyses, we observed an overall reduction of 5mC- and 5hmC-modifications at various genomic regions in OSCC. Unidirectional modification with 5mC and 5hmC at the promoters is associated with enhanced changes in gene expression in OSCC tissues. Furthermore, such differentially expressed genes bearing unidirectional modifications with 5mC and 5hmC at the promoters might have clinical relevance to the outcome of OSCC.Copyright © 2023 Zhao, Zhu, Gong, Ma, Xiong, Su, Wan and Wang.