DNA methylation is a common molecular alteration in colorectal cancer cells and culture method has no influence on DNA methylation

Abstract

The present study aimed to explore whether culture method had an influence on DNA methylation in colorectal cancer (CRC). In the present study, CRC cells were cultured in two-dimensional (2D), three-dimensional (3D) and mouse orthotopic transplantation (Tis) cultures. Principal component analysis (PCA) was used for global visualization of the three samples. A Venn diagram was applied for intersection and union analysis for different comparisons. The methylation condition of 5'-C-phosphate-G-3' (CpG) location was determined using unsupervised clustering analysis. Scatter plots and histograms of the mean β values between 3D vs. 2D, 3D vs. Tis and Tis vs. 2D were constructed. In order to explore the biological function of the genes, gene ontology and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analyses were utilized. To explore the influence of culture condition on genes, quantitative methylation specific polymerase chain reaction (QMSP) was performed. The three samples connected with each other closely, as demonstrated by PCA. Venn diagram analysis indicated that some differential methylation positions were commonly shared in the three groups of samples and 16 CpG positions appeared hypermethylated in the three samples. The methylation patterns between the 3D and 2D cultures were more similar than those of 3D and Tis, and Tis and 2D. Results of gene ontology demonstrated that differentially expressed genes were involved in molecular function, cellular components and biological function. KEGG analysis indicated that genes were enriched in 13 pathways, of which four pathways were the most evident. These pathways were pathways in cancer, mitogen-activated protein kinase signaling, axon guidance and insulin signaling. Furthermore, QMSP demonstrated that methylation of mutL homolog, phosphatase and tensin homolog, runt-related transcription factor, Ras association family member, cadherin-1, O-6-methylguanine-DNA-methyltransferase and P16 genes had no obvious difference in 2D, 3D and Tis culture conditions. In conclusion, the culture method had no influence on DNA methylation in CRC cells.

Keywords: 5′-C-phosphate-G-3′; DNA methylation; colorectal cancer; culture method.

Figure 1.

(A) Principal component analysis of dataset. The DLD1-3D, DLD1-2D and DLD1-Tis dataset were analyzed to identify the outlier of the sample group. (B) Venn diagram of differentially methylated DNA. (C) Unsupervised clustering analysis of the 5′-C-phosphate-G-3′ locations. The color gradient green to red displays the β-value and can range from 0–1. 3D, three-dimensional culture; 2D, two-dimensional culture; Tis, mouse orthotopic transplantation culture.

Figure 2.

Scatter plots indicate genome-wide methylation changes in Tamoxifen-resistant lines compared with the parental. (A) DLD1-3D vs. DLD1-2D, (B) DLD1-Tis vs. DLD1-3D and (C) DLD1-Tis vs. DVLD1-2D were compared using GenomeStudio to determine the overall changes in methylation. The black data points mark the average β cut-off value of 0.2 for each sample; center red line represents equal β values in the two samples; outer red lines mark the 2-fold change in average β values for each sample. Column distribution of β values in the (D) DLD1-2D, (E) DLD1-3D and (F) DLD1-Tis groups. 3D, three-dimensional culture; 2D, two-dimensional culture; Tis, mouse orthotopic transplantation culture.

Figure 3.

(A) Heatmap of corresponding genes at different methylation sites. (B) Significant pathways of corresponding genes at different methylation sites.

Figure 4.

Quantitative methylation specific real-time polymerase chain reaction studying the methylation condition of mutL homolog, phosphatase and tensin homolog, runt-related transcription factor, Ras association family member, cadherin-1, O-6-methylguanine-DNA-methyltransferase and P16, and related genes. (A) Association between fluorescence and cycle. (B) Rate of relaxation varies with temperature. dl/dT, rate of relaxation.