Abstract
BackgroundMethylation levels of genomic repeats such as long interspersed nucleotide elements (LINE-1) are representative of global methylation status and play an important role in maintenance of genomic stability. The objective of the study was to assess LINE-1 methylation status in colorectal cancer (CRC) in relation to adenomatous and malignant progression, tissue heterogeneity, and TNM-stage.Methodology/Principal FindingsDNA was collected by laser-capture microdissection (LCM) from normal, adenoma, and cancer tissue from 25 patients with TisN0M0 and from 92 primary CRC patients of various TNM-stages. The paraffin-embedded tissue sections were treated by in-situ DNA sodium bisulfite modification (SBM). LINE-1 hypomethylation index (LHI) was measured by absolute quantitative analysis of methylated alleles (AQAMA) realtime PCR; a greater index indicated enhanced hypomethylation. LHI in normal, cancer mesenchymal, adenoma, and CRC tissue was 0.38 (SD 0.07), 0.37 (SD 0.09), 0.49 (SD 0.10) and 0.53 (SD 0.08), respectively. LHI was significantly greater in adenoma tissue compared to its contiguous normal epithelium (P = 0.0003) and cancer mesenchymal tissue (P<0.0001). LHI did not differ significantly between adenoma and early cancer tissue of Tis stage (P = 0.20). LHI elevated with higher T-stage (P<0.04), was significantly greater in node-positive than node-negative CRC patients (P = 0.03), and was significantly greater in stage IV than all other disease stages (P<0.05).Conclusion/SignificanceBy using in-situ SBM and LCM cell selection we demonstrated early onset of LINE-1 demethylation during adenomatous change of colorectal epithelial cells and demonstrated that LINE-1 demethylation progression is linear in relation to TNM-stage progression.
Highlights
17–18% of the human genome consists of long interspersed nucleotide element (LINE-1) repeats
absolute quantitative analysis of methylated alleles (AQAMA) linearity for LINE-1 methylation level assessment First, we evaluated the accuracy of AQAMA in assessing various levels of LINE-1 methylation
The major advantage of AQAMA is that the quantitative measurement of methylated and unmethylated alleles is performed in a single PCR reaction, providing excellent control compared to two separate PCR reactions for methylated and unmethylated allele analysis
Summary
17–18% of the human genome consists of long interspersed nucleotide element (LINE-1) repeats. LINE-1 methylation status is thought to represent the genome-wide DNA methylation status, since LINE-1 sequences are highly repeated, widely interspersed human retrotransposons. 18q loss of heterozygosity (LOH) (+) colorectal cancer (CRC) show a lower mean LINE-1 methylation level [4]. An inverse relation has been reported between LINE-1 methylation and microsatellite instability (MSI)+/CpG island methylator phenotype (CIMP)+/BRAF V600E mutation CRC [5,6]. Methylation levels of genomic repeats such as long interspersed nucleotide elements (LINE-1) are representative of global methylation status and play an important role in maintenance of genomic stability. The objective of the study was to assess LINE-1 methylation status in colorectal cancer (CRC) in relation to adenomatous and malignant progression, tissue heterogeneity, and TNM-stage
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