Fig. 3

Analysis of TIMP-1 promoter methylation in breast cancer cell lines and tissues. a Schematic of the TIMP-1 promoter. The black box indicates exon1 and ‘ATG’ indicates the translational start site. Each vertical bar represents a CpG dinucleotide. The CpG island located between −157 to −32 bp includes 11 CpG sites. The arrows indicate the location of the BSP primers. b Methylation status of the TIMP-1 promoter in breast cancer cell lines. BSP analysis was conducted in 10 clones from each cell line. A solid circle represents a methylated CpG site, and an empty circle represents a non-methylated CpG site. The TNBC cell lines (MDA-231 and MDA-468) exhibited lower methylation levels compared with the non-TNBC cell lines (MCF-10A, BT474 and SK-BR-3). c Representative images of the methylation status of TIMP-1 promoter in normal tissue and in various subtypes of breast cancer tissues. d Quantification of the mean methylation levels observed in the cancer tissues. Methylation was reduced in TNBC tissues compared with the benign, luminal-A, luminal-B and HER2+ tissues (left), collectively referred to as the non-TNBC group (right). The data are presented as the mean ± SD, p < 0.05. e Expression of TIMP-1 was analyzed using RT-PCR in breast cancer cells treated with various concentrations (0–20 μM) of 5-Aza for 48 h. TIMP-1 expression was restored in BT474 cells treated with 2–20 μM of 5-Aza. In contrast, TIMP-1 expression was not restored in MDA-231 cells treated with 5-Aza due to hypomethylation of the TIMP-1 promoter