DataSheet_1_Genome-Wide Methylation Profiling of lncRNAs Reveals a Novel Progression-Related and Prognostic Marker for Colorectal Cancer.docx

Sporadic colorectal cancer (CRC) develops principally through the adenoma-carcinoma sequence. Previous studies revealed that DNA methylation alterations play a significant role in colorectal neoplastic transformation. On the other hand, long noncoding RNAs (lncRNAs) have been identified to be associated with some critical tumorigenic processes of CRC. Accumulating evidence indicates more intricate regulatory relationships between DNA methylation and lncRNAs in CRC. Nevertheless, the methylation alterations of lncRNAs at different stages of colorectal carcinogenesis based on a genome-wide scale remain elusive. Therefore, in this study, we first used an Illumina MethylationEPIC BeadChip (850K array) to identify the methylation status of lncRNAs in 12 pairs of colorectal cancerous and adjacent normal tissues from cohort I, followed by cross-validation with The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) database. Then, the abnormal hypermethylation of candidate genes in colorectal lesions was successfully confirmed by MassARRAY EpiTYPER in cohort II including 48 CRC patients, and cohort III including 286 CRC patients, 81 advanced adenoma (AA) patients and 81 nonadvanced adenoma (NAA) patients. DLX6-AS1 hypermethylation was detected at all stages of colorectal neoplasms and occurred as early as the NAA stage during colorectal neoplastic progression. The methylation levels were significantly higher in the comparisons of CRC vs. NAA (P < 0.001) and AA vs. NAA (P = 0.004). Moreover, the hypermethylation of DLX6-AS1 promoter was also found in cell-free DNA samples collected from CRC patients as compared to healthy controls (Padj = 0.003). Multivariate Cox proportional hazards regression analysis revealed DLX6-AS1 promoter hypermethylation was independently associated with poorer disease-specific survival (HR = 2.52, 95% CI: 1.35-4.69, P = 0.004) and overall survival (HR = 1.64, 95% CI: 1.02-2.64, P = 0.042) in CRC patients. Finally, a nomogram was constructed and verified by a calibration curve to predict the survival probability of individual CRC patients (C-index: 0.789). Our findings indicate DLX6-AS1 hypermethylation might be an early event during colorectal carcinogenesis and has the potential to be a novel biomarker for CRC progression and prognosis.散发性结直肠癌（sporadic colorectal cancer, CRC）主要通过腺瘤-腺癌序列（adenoma-carcinoma sequence）发生发展。既往研究表明，DNA甲基化改变在结直肠肿瘤转化过程中发挥关键作用。另一方面，长链非编码RNA（long noncoding RNAs, lncRNAs）已被证实与结直肠癌的多种核心致瘤过程密切相关。越来越多的证据显示，结直肠癌中DNA甲基化与lncRNAs之间存在更为复杂的调控关联。然而，在全基因组层面上，结直肠癌发生不同阶段的lncRNAs甲基化改变仍有待阐明。因此，本研究首先采用Illumina MethylationEPIC微珠芯片（850K芯片），对队列I中的12对结直肠癌组织及配对癌旁正常组织的lncRNAs甲基化状态进行鉴定，随后通过癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库与基因表达综合（Gene Expression Omnibus, GEO）数据库开展交叉验证。随后，本研究借助MassARRAY EpiTYPER技术，在队列II（48例CRC患者）与队列III（286例CRC患者、81例进展性腺瘤（advanced adenoma, AA）患者及81例非进展性腺瘤（nonadvanced adenoma, NAA）患者）中，成功验证了结直肠病变中候选基因的异常高甲基化状态。研究发现，DLX6-AS1高甲基化存在于结直肠肿瘤的所有阶段，且在结直肠肿瘤进展过程中最早可于NAA阶段被检测到。相较于NAA组，CRC组（P < 0.001）与AA组（P = 0.004）的DLX6-AS1甲基化水平均显著升高。此外，与健康对照相比，CRC患者的游离DNA样本中同样检测到DLX6-AS1启动子高甲基化（校正后P值=0.003）。多因素Cox比例风险回归分析显示，DLX6-AS1启动子高甲基化与CRC患者较差的疾病特异性生存率（风险比=2.52，95%置信区间：1.35-4.69，P=0.004）及总生存率（风险比=1.64，95%置信区间：1.02-2.64，P=0.042）独立相关。最后，本研究构建了列线图，并通过校准曲线对其进行验证，以预测CRC患者的个体生存概率（C指数：0.789）。本研究结果表明，DLX6-AS1高甲基化可能是结直肠癌发生过程中的早期事件，有望成为CRC进展与预后评估的新型生物标志物。