Figure 1.
Logistics of the discovery of novel genes epigenetically silenced in cervical cancer using MeDIP-on-chip.
(A) Flow chart of the experimental design. N means numbers of hypermethylated genes. SCC, Squamous cervical carcinoma; AC, Adenocarcinoma. (B) The Venn diagram shows the number of hypermethylated genes in SCC tissues, AC tissues, and normal cervical cell scrapings. Methylation enrichment occurred in 192 genes for both SCC and AC, but not normal cervixes. (C) The Venn diagram illustrates the integration of MeDIP-on-chip results with gene expression databases and tissue differential methylation regions (T-DMR) data. Genes with mRNA expression greater than 1.2 folds in normal tissues relative to cancer (N = 149), and genes within T-DMR (N = 1314) were included in the analysis [32].
Figure 2.
Confirmation of methylation status of promoters in cell lines and clinical samples.
(A) Illustrative methylation status of 31 genes, including 34 regions, in pooled normal (N pls), squamous cervical carcinoma (SCC pls), and adenocarcinoma (AC pls) samples detected by of methylation-specific PCR (MSP). Each pool contained DNA from five patients. The bluescale represents the semi-quantitative methylation levels. The gray boxes indicate unavailable results. (B) MSP results for DBC1, PDE8B, and ZNF582 in selected individual normal (N) and SCC tissues (T). M: methylation-specific primers; U: nonmethylation-specific primers. In vitro methylated DNA (IVD) was used as the positive control. (C) Bisulfite sequencing of DBC1, PED8B, and ZNF582 in tumor and normal samples. Each line indicates a single clone. Black and white circles indicate methylated and unmethylated CpG sites, respectively. The green arrows indicate the annealing regions of MSP primers. (D) The location and intensity of probes by MeDIP-on-chip. TSS represents the transcriptional start site and the arrows indicate the direction of mRNA transcription. The Y-axis shows the value from the transforming P-value (–log10) by the KS test for each probe. (E) Gene expression analysis after demethylation treatment of cervical cancer cell lines. DAZ, 5-aza-2′-deoxycytidine; TSA, trichostatin A.
Figure 3.
QMSP analysis of DBC1 and ZNF582 in cervical scrapings.
(A) Dot plots illustrate the M-index distributions. N represents the case number. P-value <0.001 was determined by the nonparametric Kruskal–Wallis rank sum test. (B) The areas under the receiver operating curves (AUCs) used to estimate accuracy. (*P-value <0.05, ***P-value <0.001; CI, confidence interval).
Table 1.
The Methylation Index (M-Index) of DBC1 and ZNF582 in the spectrum of cervical lesions.
Table 2.
The area under the ROC curve analysis for distinguishing different diagnosis groups.
Figure 4.
Representative expression of ZNF582 protein in normal and abnormal cervical tissues.
(A) Normal squamous epithelium (N = 24). (B) and (C), Cervical intraepithelial neoplasias 1 and 2 (CIN1 and CIN2, N = 22). (D) Carcinoma in situ (CIS, N = 16). (E) and (F), Squamous cervical carcinoma (SCC, N = 58) and metastatic forms (N = 7), respectively (x400). S, stroma.