Methylome, hydroxymethylome, and integrative transcriptome profiling in human CRC tissue and paired normal tissues

DNA methylation (5-mC) and hydroxymethylation (5-hmC) are regarded as important epigenetic hallmarks in the carcinogenesis of colorectal cancer by transcriptional regulation. 5hmC is an intermediate during active demethylation and maintains the equilibrium of DNA methylation. Previous studies on DNA methylation don't differentiate 5-hmC from 5-mC. Here, in order to elucidate the epigenetic mechanisms of carcinogenesis of colorectal cancer, we integrate genome wide levels of 5-mC, 5-hmC and Transcriptional expression. 12 samples, including six colorectal tumor tissues and corresponding normal colonic tissues were recruited after surgery. Genome-wide DNA methylation was determined by methylated DNA immune- precipitation sequencing (MeDIP-seq), and hydroxymethylation by hydroxyl- methylated DNA immune-precipitation sequencing (hMedip-seq). Transcriptional expression was determined by RNA-seq. Group-wise different methylation region (DMR), different hydroxyl methylation region (DhMR) and different expressed gene (DEG) were identified. Epigenetic biomarkers were screened by integrating DMR, DhMR and DEG. We found that a genome-scale distinct hydroxymethylation pattern could be used as epigenetic biomarker for clearly differentiating colorectal cancer from normal tissues. 59249 differentially methylated regions (DMR), 187172 differentially hydroxymethylated region (DhMR) and 948 differentially expressed genes (DEGs) were identified. After cross-matched genes containing DMRs or DhMRs with DEGs, seven genes were screened. Furthermore, hypermethylation of HADHB was persistently found to be correlated with its down-regulation of transcription in CRC, potentially suggesting its role as TSG. The differences of methylation, hydroxymethylation and transcriptional expression in HADHB between cancerous and normal tissues were validated among additional colorectal cancer patients. To further validate this assumption, we also performed functional analysis and found that the expression of HADHB obviously reduced cancer cells migration and invasiveness. This study provided valuable basic data for screening epigenetic biomarkers and elucidated the epigenetic mechanisms of carcinogenesis of colorectal cancer. Overall design: 12 samples, including six colorectal tumor tissues and corresponding normal colonic tissues were recruited after surgery and subjected to MeDIP-seq, hMeDIP-seq and RNA-seq.

DNA甲基化（DNA methylation, 5-mC）与羟甲基化（hydroxymethylation, 5-hmC）被认为是结直肠癌发生过程中通过转录调控发挥功能的关键表观遗传标志。5-羟甲基胞嘧啶（5-hmC）是活性去甲基化过程中的中间产物，可维持DNA甲基化的动态平衡。既往针对DNA甲基化的相关研究未对5-mC与5-hmC进行区分。为阐明结直肠癌发生的表观遗传机制，本研究整合了全基因组范围内的5-mC、5-hmC水平与转录表达谱数据。本研究共纳入12例术后样本，包含6份结直肠癌肿瘤组织及其配对的正常结肠组织。其中，全基因组DNA甲基化水平通过甲基化DNA免疫沉淀测序（methylated DNA immunoprecipitation sequencing, MeDIP-seq）测定，羟甲基化水平通过羟甲基化DNA免疫沉淀测序（hydroxymethylated DNA immunoprecipitation sequencing, hMeDIP-seq）检测，转录表达水平则经由RNA测序（RNA sequencing, RNA-seq）分析。本研究鉴定得到组间差异甲基化区域（differentially methylated region, DMR）、组间差异羟甲基化区域（differentially hydroxymethylated region, DhMR）以及差异表达基因（differentially expressed gene, DEG），并通过整合上述三类数据筛选表观遗传生物标志物。研究发现，全基因组尺度下独特的羟甲基化模式可作为表观遗传生物标志物，实现结直肠癌组织与正常组织的精准区分。本次研究共鉴定得到59249个DMR、187172个DhMR以及948个DEGs。将携带DMR或DhMR的基因与DEGs进行交叉匹配后，最终筛选得到7个目标基因。进一步分析发现，HADHB的高甲基化与其在结直肠癌（colorectal cancer, CRC）中的转录下调持续相关，提示其可能作为肿瘤抑制基因（tumor suppressor gene, TSG）发挥抑癌作用。在额外的结直肠癌患者队列中，本研究验证了HADHB的甲基化、羟甲基化以及转录表达水平在肿瘤组织与正常组织间的显著差异。为进一步验证上述假设，本研究开展了功能实验，结果表明HADHB的表达可显著抑制癌细胞的迁移与侵袭能力。本研究为结直肠癌表观遗传生物标志物的筛选提供了宝贵的基础数据，同时阐明了结直肠癌发生的表观遗传机制。实验整体设计：共纳入12例术后样本，包含6份结直肠癌肿瘤组织及其配对的正常结肠组织，所有样本均接受MeDIP-seq、hMeDIP-seq与RNA-seq检测。