H19 is a paternally imprinted gene located at 11p15.5, which encodes a non-coding transcript. Although the role of genomic imprinting in bladder cancer is not well understood, previous studies have described H19 over-expression in these tumors. It is well established that a Differentially Methylated Region (DMR) regulates its maternal monoallelic expression by acting as an insulator that precludes the binding of the transcriptional factor CTCF in the paternal allele.
Materials and methods
DNA methylation status of two distinct regions of the H19 gene was evaluated: the sixth CTCF-binding site located in the DMR (qMSP, Quantitative Real Time Methylation Specific Polymerase Chain Reaction) and the promoter-associated CpG island (MS-HRM, Methylation-Sensitive High Resolution Melting analysis) in a total of 48 tumoral samples (37 of them matched with normal adjacent tissue).
Using a pool of blood samples obtained from healthy young adults as reference to the normal imprinting, higher methylation levels of the CTCF-binding site was detected in bladder tumors compared to the normal adjacent tissue (p=0.0031). Gains of methylation were more frequently detected in non-invasive (p=0.0425) and non-recurrent (p=0.0399) papillary bladder tumors. While DNA methylation levels of H19 promoter region varied from 10 to 50% in normal adjacent bladder tissues, tumoral samples showed greater variation (10 to 100% of methylation). Heterogeneous patterns of CpG methylation were also detected in nine tumoral samples.
Our data suggest that aberrant DNA methylation is an epigenetic change potentially associated with loss of imprinting of the H19 gene in bladder cancer.