NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients

Methylation landscape and expression analysis of NDRG4 gene in breast tumor cell lines. a Schematic representation of NDRG4 genomic region, including promoter (positions ？1500 to +500 from TSS), CpG island (？556 to +869), transcription start site (TSS) and first exon. A CpG island with 82 CGs located at positions ？387 to +103 from TSS was represented in detail. b–d Percentages of methylation in these positions in two breast tumor cell lines (MCF-7 and MDA-MB231) and one melanoma cell line (MDA-MB435) before (ctrl) and 6 days after 5-aza-dC treatments (1？μM). The percentage of DNA methylation (y-axis) is shown as average C/T ratios. Graphs represent mean percent DNA methylation at each CpG and its position relative to TSS (n？=？5). e Real-time PCR analysis of NDRG4 expression in MCF-7, MDA-MB231 and MDA-MB435 before (control) and 6 days after 5-aza-dC treatments. Expression levels relative to MCF-7 control cells and normalized to GAPDH endogenous control. Error bars represent SEM of biological replicates (n？=？3). p？<？0.05 by two-tailed t tes