Urinary
miRNAs are discussed as potential
biomarkers for
bladder cancer. The majority of
miRNAs, however, are downregulated, making it difficult to utilize reduced
miRNA signals as reliable diagnostic tools. Because the downregulation of
miRNAs is frequently associated with hypermethylation of the respective regulative sequences, we studied whether
DNA hypermethylation might serve as an improved diagnostic tool compared to measuring downregulated
miRNAs.
miRNA expression arrays and individual qPCR were used to identify and confirm
miRNAs that were downregulated in malignant urothelial cells (RT4, 5637 and J82) when compared to primary, non-malignant urothelial cells (HUEPC). DNA methylation was determined by customized PCR-arrays subsequent to methylation-sensitive
DNA-restriction and by mass spectrometry.
miRNA expression and DNA methylation were determined in untreated cells and in cultures treated with the demethylating agent
5-Aza-2'-deoxycytidine. miR-200b, miR-152 and miR-10a displayed differential expression and methylation among untreated
cancer cell lines. In addition, reduced
miRNA expression of miR-200b, miR-152, and miR-10a was associated with increased DNA methylation in malignant cells versus HUEPC. Finally, the demethylation approach revealed a causal relationship between both parameters for miR-152 in 5637 and also suggests a causal connection of both parameters for miR-200b in J82 and miR-10a in 5637. In conclusion, our studies in multiple
bladder cancer cell lines and primary non-malignant urothelial cells suggest that hypermethylation of miR-152, miR-10a and miR-200b regulative DNA sequences might serve as epigenetic
bladder cancer biomarkers.