BackgroundTumor suppressor gene p16 promoter hypermethylation has been widely studied in colorectal cancer (CRC), yet its clinicopathological significance remains controversial. The methylation alterations of other regions within p16 gene are still rarely researched. The present study aimed to explore the methylation changes of p16 gene body in CRC and to find whether they were associated with clinicopathological staging of CRC.MethodsPaired colorectal cancer tissues and corresponding adjacent normal tissues from 30 CRC patients were collected. The methylation levels of two CpG islands within p16 gene body, exon 1 and exon 2, were accurately assessed simultaneously by a LC-MS/MS method. The p16 protein expressions were assessed by immunohistochemistry assay. Statistical analyses were carried out using SPSS 17.0 software. Heat-map analysis was carried out by HemI 1.0 software.ResultsIn the present study, CRC tissues showed more highly methylated than adjacent normal tissues at both CpG islands of p16 gene. And exon 2 hypermethylation was higher and more frequent than exon 1. The ROC curve analysis showed that the simultaneous use of both indicators had excellent sensitivity and specificity for distinguishing CRC tissues and adjacent normal tissues. Following, the methylation level of p16 exon 1/2 was negatively related to p16 protein expression. Further correlation analysis revealed that p16 exon 1 hypermethylation was associated with N/Dukes staging (p = 0.033), and p16 exon 2 hypermethylaiton was associated with T staging (p = 0.035).ConclusionsThe p16 gene body was remarkably hyper-methylated in CRC tissues and associated with p16 protein expression and cancer clinicopathological staging. The combination of p16 exon 1 and exon 2 could better reflect the overall methylation status of p16 gene body and provide potential biomarkers of CRC.
The identification of biomarkers would be of benefit for the diagnosis and treatment of colorectal cancer. DNA methylation in specific genomic regions, which had shown strongly association with disease genotypes, was an effective indicator to reveal the occurrence and development of cancers. To screen out methylation biomarkers for colorectal cancer (CRC), genomic DNA was isolated from colorectal cancerous and corresponding cancer-adjacent tissues collected from 30 CRC patients and then bisulfite-converted. The exon regions of 5 targeted genes (CNRIP1, HIC1, RUNX3, p15, and SFRP2) were amplified by using nested polymerase chain reaction with specific primers, and the amplicon was purified and hydrolyzed. The methylation levels of these specific regions were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed that 5 targeted exon regions were successfully amplified and confirmed by sequencing. The methodological validations indicated that LC-MS/MS was highly sensitive and accurate. The methylation levels of CNRIP1 and RUNX3 were remarkably high in CRC tissues with statistical difference when compared with corresponding cancer-adjacent individuals, while that of HIC1, p15, and SFRP2 had no difference between 2 subjects. These findings supported CNRIP1 and RUNX3 as potential DNA methylation biomarkers for CRC diagnosis and treatment, and our LC-MS/MS approach exhibited great advantages in the identification of regional DNA methylation biomarkers.
The current methods for quantifying genome-wide 5-methylcytosine (5mC) oxides are still scarce, mostly restricted with two limitations: assay sensitivity is seriously compromised with cost, assay time and sample input; epigenetic information is irreproducible during polymerase chain reaction (PCR) amplification without bisulfite pretreatment. Here, we propose a novel Polymerization Retardation Isothermal Amplification (PRIA) strategy to directly amplify the minute differences between epigenetic bases and others by arranging DNA polymerase to repetitively pass large electron-withdrawing groups tagged 5mC-oxides. We demonstrate that low abundant 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) in genomic DNA can be accurately quantified within 10 h with 100 ng sample input on a laboratory real-time quantitative PCR instrument, and even multiple samples can be analyzed simultaneously in microplates. The global levels of 5hmC and 5fC in mouse and human brain tissues, rat hippocampal neuronal tissue, mouse kidney tissue and mouse embryonic stem cells were quantified and the observations not only confirm the widespread presence of 5hmC and 5fC but also indicate their significant variation in different tissues and cells. The strategy is easily performed in almost all research and medical laboratories, and would provide the potential capability to other candidate modifications in nucleotides.
Ambiguous alteration patterns of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) involved in Alzheimer's disease (AD) obstructed the mechanism investigation of this neurological disorder from epigenetic view. Here, we applied a fully quantitative and validated LC-MS/MS method to determine genomic 5mC and 5hmC in the brain cortex of 3 month-aged (12, 15, and 18 month) AD model mouse and found significant increases of 5mC and 5hmC levels in different months of AD mouse when compared with age-matched wild-type control and exhibited rising trend from 12-month to 18-month AD mouse, thereby supporting genomic DNA methylation and hydroxymethylation were positively correlated with developing AD.
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