Global analysis of DNA methylation by Methyl-Capture sequencing reveals epigenetic control of cisplatin resistance in ovarian cancer cell

Abstract

Cisplatin resistance is one of the major reasons leading to the high death rate of ovarian cancer. Methyl-Capture sequencing (MethylCap-seq), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein with NGS, global and unbiased analysis of global DNA methylation patterns. We applied MethylCap-seq to analyze genome-wide DNA methylation profile of cisplatin sensitive ovarian cancer cell line A2780 and its isogenic derivative resistant line A2780CP. We obtained 21,763,035 raw reads for the drug resistant cell line A2780CP and 18,821,061reads for the sensitive cell line A2780. We identified 1224 hyper-methylated and 1216 hypomethylated DMRs (differentially methylated region) in A2780CP compared to A2780. Our MethylCap-seq data on this ovarian cancer cisplatin resistant model provided a good resource for the research community. We also found that A2780CP, compared to A2780, has lower observed to expected methylated CpG ratios, suggesting a lower global CpG methylation in A2780CP cells. Methylation specific PCR and bisulfite sequencing confirmed hypermethylation of PTK6, PRKCE and BCL2L1 in A2780 compared with A2780CP. Furthermore, treatment with the demethylation reagent 5-aza-dC in A2780 cells demethylated the promoters and restored the expression of PTK6, PRKCE and BCL2L1.

Figure 1. Cell survival curve of A2780 and A2780CP by the MTT assay.

Both A2780 and A2780CP were treated with cisplatin in different doses from 0 µg/ml to 16 µg/ml for 72 hours.

Figure 2. Genomic feature and CpG_o/e distribution of the hypermethylated peaks.

A. Percentage of hypermethylated peaks based on their localized genomic features. B. Distribution of CpG_o/e for hypermethylated peaks based on their localized genomic features.

Figure 3. Validation of genes with DMRs in their promoters by MS-PCR and bisulfite sequencing.

A. Real-time PCR showing the differential expression of selected genes between the cisplatin sensitive A2780 and the cisplatin resistant A2780CP cells. B. MS-PCR data showing differential methylation states in promoter regions of the selected genes between A2780 and A2780CP cells. C. Bisulfite sequencing result for promoter regions of selected gene PTK6, PRKCE and BCL2L1 (white cycle: unmethylated CpG, Black cycle: methylated CpG).

Figure 4. Restoration of gene expression of DMR affected genes by treatment with 5-aza-dc demethylation reagent and validation by MS-PCR.

A. Validation of the changes of methylation status by MS-PCR for the six genes under treatment with 0 µM and 10 µM 5-aza-dC. B. Gene expression changes (Y-axis, relative fold changes) of the selected six genes after treatment with different concentration (X-axis) of demethylation reagent 5-aza-dC.