Abstract
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 (P adj = 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.
Highlights
Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide, with an estimated 1.9 million new cases and 935,000 deaths in 2020 [1]
By DNA methylation profiling, a total of 185 differentially methylated CpG sites mapping to the promoter of lncRNAs were identified by the 850K array generated from 12 pairs of colorectal cancerous and adjacent normal tissues, followed by cross-validation using DNA methylation data generated by the 450K array in CRCs from the The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database, respectively (Supplementary Table S4)
Among the list of CpG sites, we focused on six sites ranking on the top for following technical confirmation analysis (Figure 3), which were considered as candidate biomarkers
Summary
Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide, with an estimated 1.9 million new cases and 935,000 deaths in 2020 [1]. Many studies have reported DNA methylation changes in cancer-related genes in CRC using genome-wide-based approaches or candidate gene strategies [6–8]. These aberrant methylation alterations occur more frequently at the early stages of neoplastic progression [6]. Hierarchical hypermethylation patterns of CRC-related suppressor genes, such as SFRP2, SEPT9 and MPPED2, have been observed throughout the progression stages of colorectal carcinogenesis [9–11]. Taken together, these findings indicate that abnormal changes in DNA methylation might be hallmarks of CRC initiation and progression. DNA hypermethylation might be one of the first detectable neoplastic alterations associated with carcinogenesis
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