Intragenic DNA Methylation Concomitant with Repression of ATP4B and ATP4A Gene Expression in Gastric Cancer is a Potential Serum Biomarker

Abstract

Based on our previous report on gastric cancer which documented ATP4A and ATP4B mRNA downregulationin gastric tumors relative to normal gastric tissues, we hypothesized that epigenetic mechanisms couldbe responsible. ATP4A and ATP4B mRNA expression in gastric cancer cell lines AGS, SNU638 and NUGC-3was examined using reverse transcriptase PCR (RT-PCR). AGS cells were treated with TSA or 5’-AzaDC andmethylation specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis were performed. MSP analysiswas on DNA from paraffin embedded tissues sections and plasma. Expression analysis revealed downregulationof ATP4A and ATP4B genes in gastric cancer cell lines relative to normal gastric tissue, while treatment with5’-AzaDC re-activated expression of both. Search for CpG islands in their putative promoter regions did notindicate CpG islands (CGI) but only further downstream in the bodies of the genes. Methylation specific PCR(MSP) in the exon1 of the ATP4B gene and exon7 in ATP4A indicated methylation in all the gastric cancercell lines tested. MSP analysis in tumor tissue samples revealed methylation in the majority of tumor samples,15/19, for ATP4B and 8/8 for ATP4A. There was concordance between ATP4B and ATP4A down-regulationand methylation status in the tumour samples tested. ATP4B methylation was detectable in cell free DNA fromgastric cancer patient’s plasma samples. Thus ATP4A and ATP4B down-regulation involves DNA methylationand methylated ATP4B DNA in plasma is a potential biomarker for gastric cancer.