Endothelial PAS domain protein 1 gene hypomethylation is associated with colorectal cancer in Han Chinese

Abstract

Endothelial PAS domain-containing protein 1 (EPAS1) serves a role in angiogenesis, which is important for the development of tumors, including colorectal cancer (CRC). The current study aimed to estimate whether EPAS1 methylation was associated with CRC. A two-stage association study of EPAS1 methylation and CRC was conducted. In the first phase, EPAS1 methylation was evaluated in the tumor and adjacent non-tumor tissue samples from 41 patients with sporadic CRC in Jiangsu province, China. The diagnostic value of methylation of EPAS1 for CRC in the second phase was evaluated in 79 patients with sporadic CRC and 22 normal individuals in Zhejiang province, China. The methylation assay was performed using a quantitative methylation-specific polymerase chain reaction (qMSP) method. The percentage of methylated reference (PMR) was used to quantify the methylation level. The first-stage results indicated that EPAS1 promoter methylation was significantly lower in CRC tumor tissues compared with 5-cm-para-tumor tissues (median PMR, 0.59 vs. 1.22%; P=0.027) and 10-cm-para-tumor tissues (median PMR, 0.59 vs. 1.89%; P=0.001). In addition, the second-stage results indicated that EPAS1 promoter methylation was significantly lower in tumor tissues compared with 5-cm-para-tumor tissues (median PMR, 1.91 vs. 6.25%; P=3×10^-7) and normal intestinal tissues from healthy controls (median PMR, 1.91 vs. 28.4%; P=5×10^-7). Receiver Operating Characteristic curve analysis of the second-stage data indicated that the highest area under the curve of EPAS1 hypomethylation was 0.851 between Zhejiang CRC tissues and Zhejiang normal intestinal tissues (sensitivity, 95.5%; specificity, 60.8%).

Keywords: DNA methylation; colorectal cancer; endothelial PAS domain-containing protein 1; quantitative methylation-specific polymerase chain reaction.

Figure 1.

Characteristics of the target sequence in EPAS1. (A) Target sequences in EPAS1 promoter region. The genomic position of the amplified fragment from University of California Santa Cruz genome browser according to human (GRCh37) assembly. The primers are underlined and two CpG sites are presented in bold fond with grey highlight. (B) Capillary electrophoresis for the amplified fragment (95 bp). (C) Sanger sequencing results. The top row of the sequence is the original sequence of the fragment. The bottom row of the sequence is the converted sequence; CG dinucleotides that remained unaltered are in blue boxes; and C nucleotides with corresponding converted T nucleotides are in black boxes. F, forward primer; R, reverse primer; EPAS1, endothelial PAS domain-containing protein 1; CpG, cytosine-phosphate-guanine.

Figure 2.

Comparisons of EPAS1 methylation levels between patients and normal controls. (A) Comparisons of methylation levels between tumor tissues, 5-cm-para-tumor tissues and 10-cm-para-tumor tissues (Jiangsu, China). (B) Comparisons of methylation levels between tumor tissues, 5-cm-para-tumor tissues and normal intestinal tissues of healthy controls (Zhejiang, China). EPAS1, endothelial PAS domain-containing protein 1; PMR, percentage of methylated reference.

Figure 3.

Bioinformatics analysis of the correlation between EPAS1 methylation and expression. An inverse correlation was identified between EPAS1 methylation and mRNA expression in 372 colorectal cancer samples (r=−0.329, P=8×10⁻¹¹). EPAS1, endothelial PAS domain-containing protein 1.

Figure 4.

ROC curves for the diagnostic value of EPAS1 hypomethylation in the second-stage experiment. (A) ROC curve for the diagnostic value of EPAS1 hypomethylation between CRC tissues and paired non-cancer tissues (Zhejiang, China). (B) ROC curve for the diagnostic value of EPAS1 hypomethylation between CRC tissues and normal intestinal tissues of healthy controls (Zhejiang, China). (C) ROC curve for the diagnostic value of EPAS1 hypomethylation between paired non-cancer tissues and normal intestinal tissues of healthy controls (Zhejiang, China). ROC, receiver operating characteristic; AUC, area under the curve; EPAS1, endothelial PAS domain-containing protein 1; CRC, colorectal cancer.