DNA methylation profiling revealed promoter hypermethylation-induced silencing of p16, DDAH2 and DUSP1 in primary oral squamous cell carcinoma

Abstract

Background: Hypermethylation in promoter regions of genes might lead to altered gene functions and result in malignant cellular transformation. Thus, biomarker identification for hypermethylated genes would be very useful for early diagnosis, prognosis, and therapeutic treatment of oral squamous cell carcinoma (OSCC). The objectives of this study were to screen and validate differentially hypermethylated genes in OSCC and correlate the hypermethylation-induced genes with demographic, clinocopathological characteristics and survival rate of OSCC.

Methods: DNA methylation profiling was utilized to screen the differentially hypermethylated genes in OSCC. Three selected differentially-hypermethylated genes of p16, DDAH2 and DUSP1 were further validated for methylation status and protein expression. The correlation between demographic, clinicopathological characteristics, and survival rate of OSCC patients with hypermethylation of p16, DDAH2 and DUSP1 genes were analysed in the study.

Results: Methylation profiling demonstrated 33 promoter hypermethylated genes in OSCC. The differentially-hypermethylated genes of p16, DDAH2 and DUSP1 revealed positivity of 78%, 80% and 88% in methylation-specific polymerase chain reaction and 24% and 22% of immunoreactivity in DDAH2 and DUSP1 genes, respectively. Promoter hypermethylation of p16 gene was found significantly associated with tumour site of buccal, gum, tongue and lip (P=0.001). In addition, DDAH2 methylation level was correlated significantly with patients' age (P=0.050). In this study, overall five-year survival rate was 38.1% for OSCC patients and was influenced by sex difference.

Conclusions: The study has identified 33 promoter hypermethylated genes that were significantly silenced in OSCC, which might be involved in an important mechanism in oral carcinogenesis. Our approaches revealed signature candidates of differentially hypermethylated genes of DDAH2 and DUSP1 which can be further developed as potential biomarkers for OSCC as diagnostic, prognostic and therapeutic targets in the future.

Keywords: DDAH2; DNA methylation profiling; DUSP1; Oral Squamous Cell Carcinoma; promoter hypermethylation; protein expression..

Fig 1

Histogram of group methylation profiles of (a) p16 (b) DDAH2 and (c) DUSP1 alleles average β value between normal and 4 pathological stages (Stage 1, 2, 3 and 4). Distinct profile shows lower average β value in normal subjects if compared with pathological stage 1, 2, 3 and 4 for (a) p16 (b) DDAH2 and (c) DUSP1 alleles.

Fig 2

Heatmap methylation frequency of differentially methylated genes in OSCC. Unsupervised hierarchical clustering was performed on gene methylation profiles for the normal tissues (N=3) and tumour tissues (N=20, Stage 1, 2, 3 and 4). The heat map of differentially methylated genes based on clustering is shown in the figure. Each row represents a sample and each column represents a CpG loci. Red colour indicates hypermethylated CpG sites and blue colour indicates hypomethylated CpG sites.

Fig 3

Partek Genomic Suite Visualization. The upper panel of each shows the heat map for each probe of (a) p16, (b) DUSP1 and (c) DDAH2 genes in normal and four pathological stages. The line graphs in the lower panel of each show log2 ratio of β values of each probe between normal and four pathological stages with clear separation (indicated with red arrow).

Fig 3

Partek Genomic Suite Visualization. The upper panel of each shows the heat map for each probe of (a) p16, (b) DUSP1 and (c) DDAH2 genes in normal and four pathological stages. The line graphs in the lower panel of each show log2 ratio of β values of each probe between normal and four pathological stages with clear separation (indicated with red arrow).

Fig 3

Partek Genomic Suite Visualization. The upper panel of each shows the heat map for each probe of (a) p16, (b) DUSP1 and (c) DDAH2 genes in normal and four pathological stages. The line graphs in the lower panel of each show log2 ratio of β values of each probe between normal and four pathological stages with clear separation (indicated with red arrow).

Fig 4

Three representative agarose gel electrophoretic images of hypermethylation status for genes of (a) p16, (b) DDAH2 and (c) DUSP1 in methylation control and tumour samples. DKO represents methylation control; M represents methylated alleles and U represents unmethylated alleles. DKO controls (DKO M and DKO U) show bands on each gel and each tumour samples show methylated status of p16 (a), DDAH2 (b), and DUSP1 (c).

Fig 5

Formaline-fixed paraffin-embedded sections of representative Oral Squamous Cell Carcinoma tissues were stained with the antibodies against DDAH2 and DUSP1. (a) Negative immunostaining of DDAH2 in nuclei of the tumour cells. (b) Positive staining of DDAH2 was detected in nuclei of the tumour cells. (c) Negative cytoplasmic immunostaining of DUSP1 in the tumour cells. (d) Positive DUSP1 cytoplasmic staining was detected in the tumour cells.

Fig 6

Kaplan-Meier survival curve shows 38.1% of overall five-year survival rate.

Fig 7

Kaplan-Meier survival curve shows sex difference influences differently on OSCC patients' overall five-year survival rate.