Methylation-specific high-resolution melting analysis (MS-HRM)-based DNA promoter profiling in paired FFPE bladder cancer samples.

Abstract

315 Background: DNA methylation and histone modification are widely studied epigenetic events that can decrease gene expression levels. Promoter hypermethylation has been proposed as a potential diagnostic or prognostic biomarker in various cancers. We studied DNA promoter methylation of 5 cancer-associated genes (MRE11, APX1, ERCC1, RASSF1A and RASSF2A ) in paired FFPE bladder cancer tissues and normal adjacent tissue. The MRE11/Rad50/NBS1 complex serves as a single-strand DNA nuclease which participates in the repair of DNA double-strand breaks and replication errors. APX1 plays a key role in regulating H2O2 levels and H2O2 signaling. DNA repair machinery, ERCC1 protein levels in tumor tissue have been shown to predict response to platinum-based chemotherapy. RASSF1 is thought to be a tumor suppressor gene, while the function of RASSF2 is less well understood. Methods: 16 bladder cancer cases with available paraffin-embedded tumor and matched normal adjacent tissue specimens (32 tissue samples) were analyzed. DNA was extracted by Ambion RecoverAll Total Nucleic Acid Isolation kit. FFPE DNA bisulfite modification was performed using Zymo EZ DNA Methylation Kit. Methylation Specific-High Resolution Melting (MS-HRM) analysis was used to assess methylation status. MS-HRM monitors the melting behavior of PCR amplicons by using a DNA intercalating fluorescent dye. Results: MRE11 and APX1 promoters were not methylated in either cancer or normal adjacent samples. The ERCC1 gene was heavily methylated in 94% (30/32) of all cancer and wild type samples. RASSF1A and RASSF2A promoter methylation was significantly different between cancer and normal adjacent tissue. 50% (8/16) RASSF1A and 25% (4/16) RASSF2A had promoter methylation in cancer tissues, while only 6% (1/16) RASSF1A and 0% (0/16) RASSF2A had promoter methylation in normal adjacent tissues. 69% (11/16) of bladder cancer tissues were positive for RASSF1A or RASSF2A promotor methylation while only 1/16 normal adjacent tissue samples was positive for either promotor methylation. Conclusions: The results show that cancer and non-cancer tissue have different RASSF1A and RASSF2A promoter methylation patterns.