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Published on: October 18, 2013
Gene promoter and exon DNA methylation changes in colon cancer development - mRNA expression and tumor mutation
Béla Molnár1,2, Orsolya Galamb3, Bálint Péterfia4
1Molecular Medicine Research Group, Hungarian Academy of Sciences, Szentkirályi str 46, Budapest, H-1088, Hungary. molnar.bela1@med.semmelweis-univ.hu.
Background:
DNA mutations occur randomly and sporadically in growth-related genes, mostly on cytosines. Demethylation of cytosines may lead to genetic instability through spontaneous deamination. Aims were whole genome methylation and targeted mutation analysis of colorectal cancer (CRC)-related genes and mRNA expression analysis of TP53 pathway genes.
Methods:
Long interspersed nuclear element-1 (LINE-1) BS-PCR followed by pyrosequencing was performed for the estimation of global DNA metlyation levels along the colorectal normal-adenoma-carcinoma sequence. Methyl capture sequencing was done on 6 normal adjacent (NAT), 15 adenomatous (AD) and 9 CRC tissues. Overall quantitative methylation analysis, selection of top hyper/hypomethylated genes, methylation analysis on mutation regions and TP53 pathway gene promoters were performed. Mutations of 12 CRC-related genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53) were evaluated. mRNA expression of TP53 pathway genes was also analyzed.
Results:
According to the LINE-1 methylation results, overall hypomethylation was observed along the normal-adenoma-carcinoma sequence. Within top50 differential methylated regions (DMRs), in AD-N comparison TP73, NGFR, PDGFRA genes were hypermethylated, FMN1, SLC16A7 genes were hypomethylated. In CRC-N comparison DKK2, SDC2, SOX1 genes showed hypermethylation, while ERBB4, CREB5, CNTN1 genes were hypomethylated. In certain mutation hot spot regions significant DNA methylation alterations were detected. The TP53 gene body was addressed by hypermethylation in adenomas. APC, TP53 and KRAS mutations were found in 30, 15, 21% of adenomas, and in 29, 53, 29% of CRCs, respectively. mRNA expression changes were observed in several TP53 pathway genes showing promoter methylation alterations.
Conclusions:
DNA methylation with consecutive phenotypic effect can be observed in a high number of promoter and gene body regions through CRC development.
Insights
Colorectal cancer (CRC) development involves widespread DNA methylation changes, particularly hypomethylation of LINE-1 elements and alterations in specific gene promoters. These epigenetic modifications correlate with mutations and gene expression changes in key cancer pathways.
Area of Science:
- Molecular biology
- Cancer genetics
- Epigenetics
Background:
- DNA mutations are random, often affecting cytosines, and demethylation can cause instability.
- Spontaneous deamination of cytosines is a source of genetic instability.
- Colorectal cancer (CRC) development involves complex genetic and epigenetic alterations.
Purpose of the Study:
- To analyze whole genome methylation patterns in colorectal cancer (CRC).
- To perform targeted mutation analysis of CRC-related genes.
- To investigate mRNA expression of TP53 pathway genes in relation to methylation.
Main Methods:
- Global DNA methylation was assessed using Long interspersed nuclear element-1 (LINE-1) bisulfite PCR and pyrosequencing.
- Methyl capture sequencing was performed on normal adjacent, adenomatous, and CRC tissues.
- Mutations in 12 CRC-related genes and mRNA expression of TP53 pathway genes were analyzed.
Main Results:
- Overall hypomethylation was observed across the normal-adenoma-carcinoma sequence.
- Differential methylation occurred in genes like TP73, NGFR, PDGFRA, FMN1, SLC16A7, DKK2, SDC2, SOX1, ERBB4, CREB5, and CNTN1.
- Significant DNA methylation alterations were found in mutation hotspots, with TP53 gene body hypermethylation in adenomas. APC, TP53, and KRAS mutations were frequent.
Conclusions:
- DNA methylation changes significantly impact gene promoter and body regions during colorectal cancer progression.
- Epigenetic alterations contribute to the phenotypic effects observed in CRC development.
- Methylation patterns are closely linked to genetic mutations and gene expression in CRC.
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