The hypermethylation of p16 gene exon 1 and exon 2: potential biomarkers for colorectal cancer and are associated with cancer pathological staging

Abstract

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.

High‐glucose concentration aggravates TNF‐alpha‐induced cell viability reduction in human CD146‐positive periodontal ligament cells via TNFR‐1 gene demethylation

“…However, gene body methylation is linked to the recruitment of transcriptional repressors such as methyl‐CpG‐binding protein 2 (Bird, 2002) and the reduction of transcriptional elongation by reducing RNA polymerase II binding efficiency (Lorincz, Dickerson, Schmitt, & Groudine, 2004). Moreover, the majority of the evidence indicated that gene body hypermethylation resulted in lower gene expression (Manjegowda, Gupta, & Limaye, 2017; Matsubara et al, 2010; Oda et al, 2018; Xiaoxia, et al, 2018).…”

Suppressor genes as molecular markers of colorectal cancer – a review of the latest reports

“…In addition, a positive correlation between the degree of methylation and the stage of cancer was observed. These data indicate that the studied regions of the p16 gene may become new, more specific CRC diagnostic biomarkers and the target of new therapies [19].…”

“…It has been proven that the main mechanism leading to the inactivation of the p16 gene is hypermethylation, which is a common phenomenon in colorectal cancer [17,18]. Literature data from 2018 indicate that studies on p16 gene methylated regions in relation to the stage of cancer are underway [19]. To date, the association between hypermethylation of p16 gene regions and p16 protein expression has remained controversial, mainly due to inconsistent, incomparable results obtained by methods that test shorter fragments, different CpG regions, or other gene target sites.…”

MafF Is Regulated via the circ-ITCH/miR-224-5p Axis and Acts as a Tumor Suppressor in Hepatocellular Carcinoma

“…In each experiment, the primary antibody was omitted as a negative control. The sections were evaluated independently by two investigators evaluated independently according to our previous report with slight modification 23 . The degree of immunohistochemical staining was assessed by multiplying the staining intensity score (0: no, 1: light yellow, 2: yellow, and 3: brown yellow) and the staining proportion score (0: < 25%, 1: 25%–50%, 2: 51%–75%, and 3: >75%).…”