DNA methylation profile at the DNMT3L promoter: a potential biomarker for cervical cancer

Abstract

Epigenetic events play a prominent role during cancer development. This is evident from the fact that almost all cancer types show aberrant DNA methylation. These abnormal DNA methylation levels are not restricted to just a few genes but affect the whole genome. Previous studies have shown genome-wide DNA hypomethylation and gene-specific hypermethylation to be a hallmark of most cancers. Molecules like DNA methyltransferase act as effectors of epigenetic reprogramming. In the present study we have examined the possibility that the reprogramming genes themselves undergo epigenetic modifications reflecting their changed transcriptional status during cancer development. Comparison of DNA methylation status between the normal and cervical cancer samples was carried out at the promoters of a few reprogramming molecules. Our study revealed statistically significant DNA methylation differences within the promoter of DNMT3L. A regulator of de novo DNA methyltransferases DNMT3A and DNMT3B, DNMT3L promoter was found to have lost DNA methylation to varying levels in 14 out of 15 cancer cervix samples analysed. The present study highlights the importance of DNA methylation profile at DNMT3L promoter not only as a promising biomarker for cervical cancer, which is the second most common cancer among women worldwide, but also provides insight into the possible role of DNMT3L in cancer development.

Figure 1

Comparative analysis of DNA methylation at the RASSF1A promoter. Bisulfite sequencing analysis was performed on DNA isolated from normal and cancer cervix patients. Each horizontal line indicates a single clone from the respective PCR products after bisulfite treatment. Circles denote CpG dinucleotides present within the sequence. The positions are not drawn to scale. Open circles indicate no methylation. Filled circles represent methylated cytosine. Each bracketed profile represents individual sample. Normal cancer cervix samples are prefixed with NC and cancer samples have CC as a prefix.

Figure 2

Summary of DNA methylation results on the 9 genes analysed. Each colored box represents one CpG dinucleotide. Each gene is represented by colored boxes equal to the number of CpG analysed. Respective color denotes the percentage of clones showing methylation at individual CpG dinucleotide. Green: 0-34%, Yellow: 34-66%, Red: 66-100%. Note the remarkable difference in the methylation levels for DNMT3L between normal and cancer cervix samples (except CC2). Symbols below each CpG box for DNMT3L denotes statistical significant difference (calculated using t-test) in methylation for the respective CpG between normal and cancer samples (+ - p<0.05, * - p<0.001). No correlation was found between the grade of tumor and changes in DNA methylation levels. The sequences of the primers designed for bisulfite PCR using Methprimer are provided in (Supplementary Table T1).

Figure 3

Comparative analysis of DNA methylation at the DNMT3L promoter. Bisulfite sequencing analysis was performed on DNA isolated from normal and cancer cervix patients as described in materials and methods. See (Fig. 1) legend for explanation of the figure.

Figure 4

DNMT3L promoter methylation is correlated to its expression. (A) Bisulfite analysis of DNMT3L promoter for HeLa, and SiHa cell lines. (B) Real-time QRT-PCR analysis for DNMT3L mRNA extracted from SiHa and Hela cervical cancer cell lines. Results are shown as fold difference in the DNMT3L expression levels. Each experiment was done in triplicate and was repeated thrice and normalized against the amount of b-ACTIN mRNA in each sample.