Non-CpG methylation of Oct4 genomic elements and the CpG methylation pattern of the PSA promoter make it possible to distinguish between aggressive and non-aggressive PCa in a model of cell lines

The purpose of the study is to identify the distinctive 5-mC patterns of Oct4 and PSA genomic elements using DNA of PC3 and LNCaP prostate cancer-derived cell lines and BPH-1 and HPrEC as non-cancerous control. All cell lines were cultured at 37◦ C in a 5% CO2 atmosphere incubator according to company-provided protocol. The bisulfite treated DNA was used to amplify the genomic region of the Oct4 and PSA genes with primers specific for bisulfite converted DNA. 10-12 clones for each PCR product were sequenced and analysed. DNA sequencing was performed using the ABI BigDyeTerminator Cycle Sequencing Kit v3 and the sequences were detected on an ABI 3130XL Genetic Analyzer. Only CpG methylation of Oct4 promoter region was found in HPrEC cell line. All CpG analysed of promoter region was methylated. In opposite, the cancerous PC3 and LNCaP cell lines and the benign prostatic BPH1 cell line in addition to CpG methylation had a CCWGG and WCWGG pentanucleotide methylation, where W=A/T. In respect to WCWGG this is new observation. We found that known CCWGG methylation predominantly distributed in PC3 cell line but WCWGG is more characteristic for BPH and LNCaP cell lines. Also, we demonstrated that the promoter region of PSA in cell line studied had the distinctive methylation pattern. The CpGs of PSA promoter in LNCaP cells were free of methylation, while PC3 cells showed fully methylated PSA promoter spanning region. Using the found differential methylation patterns we have designed the primers and developed the nested methylation specific PCR (MS-PCR) to distinguish between an aggressive and non-aggressive PCa in a model of cell lines. The developed nested MS-PCR makes it possible to distinguish between an aggressive and non-aggressive PCa in a model of cell lines.