Unraveling TNXB Epigenetic Alterations Through Genome-Wide DNA Methylation Analysis and Their Implications for Colorectal Cancer

Abstract

Aberrant DNA methylation has been shown to be a fingerprint characteristic in human colorectal tumors. In this study, we hypothesize that investigating global DNA methylation could offer potential candidates for clinical application in CRC. The epigenome-wide association analysis was conducted in both the tumor area (N = 27) and the adjacent tumor-free (NAT) area (N = 15). We found 78,935 differentially methylated CpG sites (DMCs) (FDR < 0.05), 42,888 hypomethylated and 36,047 hypermethylation showing overall hypomethylation. Gene ontology and KEGG analysis of differentially methylated genes showed significant enrichment in developmental genes, as well as in genes involved in metabolic processes and the cell cycle, such as the TFGβ and cAMP signaling pathways. Through filtered analysis, we identified TNXB as the most epigenetically dysregulated gene, hypomethylated and downregulated in CRC (both with p < 0.001) and associated with poor overall survival. In the functional analysis, TNXB was epigenetically regulated in a dose-dependent manner, suggesting a potential role in CRC. The epigenetic dysregulation and functional role of TNXB in CRC could have clinical implications, serving as indicators of malignant potential, with adverse effects associated with disease origin and progression in CRC.

Keywords: 450K; DNA methylation; TNXB; colorectal cancer; epigenetic.

Figure 1

Profile of DNA methylation in tumors from colorectal cancer patients. (A) Principal component analysis (PCA) for DNA methylation levels of the 1000 most variable CpGs between the tumor area and NAT area, showing two well-clustered groups. (B) Comparison between global DNA methylation and global promoter methylation and the tumor area and the NAT area. Asterisks indicate significant differences between the groups according to the Mann–Whitney test (*** p < 0.001). (C) Miami plot showing the y axis with the Log₁₀(FDR) values of CpGs, and the x axis shows their chromosomal position. This plot displays Log₁₀(FDR) for DMC associations for hypermethylated CpG sites in the upper part, and for hypomethylated CpG sites in the lower part. Blue line indicates the genome-wide significance level (p < Log₁₀(FDR). (D) After a stringent analysis with an FDR < 0.001, we found a total of 73,509 DMCs. After selecting by a LogFC > |1.2|, we found 5124 hypermethylated probes and 3578 hypomethylated CpGs. Genomic distribution of the DMCs and their respective locations regarding the broader CpG context over the genome region. (E,F) Gene Set Enrichment Analysis gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, including the most significant DMCs. Abbreviations: DMCs: differentially methylated CpG; FDR: false discovery rate; IGR: intergenic region; NAT: normal adjacent tumor; LogFC: log fold change; TSS: transcription site start; UTR: untranslated region; DMR: differentially methylated regions, (C) cancer (R) reprogramming specific.

Figure 2

Validation analysis of TNXB. (A) β methylation of the TNXB gene was extracted from the DNA array. We specifically selected DMC sites located at the TNXB gene from both tumor and NAT samples. Asterisks indicate significant differences between the groups according to the Mann–Whitney test (** p < 0.01, *** p < 0.001). Normalized gene expression of TNXB in the tumor (N = 30) and NAT (N = 24) areas. Gene expression was normalized using the PPIA gene, and using the following formula: 2^−ΔCt. Asterisks indicate significant differences between the groups according to the Mann–Whitney test (** p < 0.01, *** p < 0.001). Gene expression (extracted from RNA-seq analysis) and methylation of the TNXB gene extracted from (B) TCGA-COAD and (C) TCGA-READ, and Asterisks indicate significant differences between the groups according to the Mann–Whitney test (*** p < 0.001). (D) A representation of the total of DMCs and DMRs located at the TNXB gene. Representation of DMR 1 and DMR 5 found in our study and belonging to the TNXB gene. (E) Correlation analysis using Spearman’s correlation between TNXB methylation and expression (N = 8). (F) Kaplan–Meier analysis comparing low (lowest and middle tertiles; N = 20) and high (highest tertile; N = 7) methylation analysis.

Figure 3

Epigenetic dysregulation of TNXB in colorectal cancer cell line HCT116. HCT116 cells were treated with 5-Aza-2′-deoxycytidine (AZA) for 72 h to induce DNA demethylation. TNXB gene expression was measured by quantitative RT-PCR. The results indicate that epigenetic silencing via DNA methylation regulates TNXB expression in colorectal cancer cells. Abbreviations: AZA, 5-Aza-2′-deoxycytidine; TNXB, Tenascin XB. *** p < 0.001.