Abstract

Background
We have recently profiled the expression of microRNA in a large bladder cancer (BC) cohort. We found malignant loss and gain of expression for numerous solitary and clustered microRNAs located within proximity of CpG islands. As aberrant hypermethylation of microRNA promoter regions has been described in other tumours we sought to systematically examine this epigenetic trait in BC.

Method
From UCSC, Ensembl and miRBase we constructed a bioinformatic database of all human microRNAs with respect to gene loci, location, clustering and CpG islands. Using an Agilent oligonucleotide microarray we profiled the expression of 985 microRNAs in 4 urothelial cells lines before and after DNA methyl-transferase inhibition with 5-azacytidine, in triplicate. We confirmed altered expression with QrtPCR for 365 miRs (Taqman). The cells were chosen to reflect normal urothelium (NHU) and well (RT4), moderate (RT112) and poorly differentiated BC. We bisulphite sequenced the CpG Island and Shore regions of microRNA with differential expression between the treated and control cells. Those with differential methylation were examined in a cohort of 100 BC using methyl specific PCR.

Results
Around 13% and 28% of human microRNA are located with 3kb and 10kb of a CpG island, respectively. 5-Azacytine treatment leads to altered expression of many miRs and for 76 this pattern occurred in more than one cell line. Microarray expression and realtime PCR were closely correlated (r=-0.65 to -0.9, p<0.0001). Bisulphite sequencing revealed tumour specific methylation of microRNAs-9-1/-2/-3, microRNA-1224 and those around CpG island 63 on Chromosome 19. Methyl specific PCR revealed frequent methylation of these microRNA in primary bladder cancers and various associations with phenotype.

Conclusion
Epigenetic regulation of microRNA is associated with carcinogenic alterations in BC. Specifically, silencing of microRNAs-9/1224 and those on chromosome 19 may contribute to disease pathogenesis and could be therapeutic targets or predictive biomarkers. The implications of silencing require functional evaluation.