Abstract
Research on DNA methylation offers great potential for the identification of biomarkers that can be applied for accurately assessing an individual's risk for cancer. In this article, we try to find the ideal epigenetic genes involved in colorectal cancer (CRC) based on a CRC database and our CRC cohort. The top 20 genes with an extremely high frequency of hypermethylation in CRC were identified in the latest database. Remarkably, 3 HOXA genes were included in this list and ranked at the top. The percentage of methylation in the HOXA5, HOXA2, and HOXA6 genes in CRC were up to 67.62, 58.36, and 31.32%, respectively, and ranked first in CRC among all human tumor tissues. Paired colorectal tumor samples and adjacent non-tumor colorectal tissue samples and four CRC cell lines were selected for MethylTarget™ assays. The results demonstrated that CRC tissues and cells had a stronger methylation status around the 3 HOXA gene promoter regions compared with adjacent non-tumor colonic tissue samples. The Receiver operator characteristic curve (ROC) curves for HOXA genes show excellent diagnostic ability in distinguishing tissue from healthy individuals and CRC patients, especially for Stage I patients (AUC = 0.9979 in HOXA2, 0.9309 in HOXA5, and 0.8025 in HOXA6). An association analysis between the methylation pattern of HOXA genes and clinical indicators was performed and found that HOXA2 methylation was significantly associated with age, N, stage, M, lymphovascular invasion, perineural invasion, lymph node number. HOXA5 methylation was associated with age, T, M, stage, and tumor status, and HOXA6 methylation was associated with age and KRAS mutation. Notably, we found that the highest methylation of HOXA5 and HOXA2 occurs in the early stages of colorectal cancer tissues such as stage I, N0, MO, and non-invasive tissues. The methylation levels declined as tumors progressed. However, methylation level at any stage of the tumor was still significantly higher than in normal tissues (p < 0.0001). The mRNA of the 3 HOXA genes was downregulated in early tumor stages due to hypermethylation of CpG islands adjacent to the promoters of the genes. In addition, hypermethylation of HOXA5 and HOXA6 mainly occurred in patients < 60 years old and with MSI-L, MSS, CIMP.L and non-CIMP tumors. Together, this suggests that epigenetic silencing of 3 adjacent HOXA genes may be an important event in the progression of colorectal cancer.
Highlights
Colorectal cancer (CRC) is a critical public health problem as it is the fourth most common cancer diagnosed among adults and the second leading cause of death from cancer in the developed world, and the CRC death rate is still increasing in many developing countries [1, 2]
We found that the methylation percentages of HOXA5, HOXA2, and HOXA6 in CRC tissue were up to 67.62, 58.36, and 31.32%, respectively, and ranked first in CRC among all cancer types analyzed (Figure 1B)
The DNA methylation profiles of the 3 HOXA gene promoter regions were downloaded from TCGA, and the results demonstrated that the methylation levels of HOXA5, HOXA2, and HOXA6 were significantly elevated in cancer tissues compared with normal tissues (p < 0.0001) (Figures 1C–E)
Summary
Colorectal cancer (CRC) is a critical public health problem as it is the fourth most common cancer diagnosed among adults and the second leading cause of death from cancer in the developed world, and the CRC death rate is still increasing in many developing countries [1, 2]. We found that the genes located on chromosome 20q were frequently upregulated in colorectal cancer, and these genes can be studied as optimal candidates for oncogenes. We understand that tumor suppressor gene silencing by promoter hypermethylation is an important mechanism in carcinogenesis [7, 8]. There are numerous examples of this mechanism in colorectal cancer, such as the mismatch repair gene human mutL homolog 1 (MLH1) with notable methylation and aberrant patterns of DNA methylation leading to activation of the Wnt pathway by silencing the secreted frizzled-related protein (SFRP) family of genes [9, 10]. In this article, based on the latest colorectal cancer database and our experiments, we analyze the methylation status in three HOXA genes including HOXA2, HOXA5, and HOXA6 and its clinical application
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