A systematic analysis on DNA methylation and the expression of both mRNA and microRNA in bladder cancer

Abstract

Background: DNA methylation aberration and microRNA (miRNA) deregulation have been observed in many types of cancers. A systematic study of methylome and transcriptome in bladder urothelial carcinoma has never been reported.

Methodology/principal findings: The DNA methylation was profiled by modified methylation-specific digital karyotyping (MMSDK) and the expression of mRNAs and miRNAs was analyzed by digital gene expression (DGE) sequencing in tumors and matched normal adjacent tissues obtained from 9 bladder urothelial carcinoma patients. We found that a set of significantly enriched pathways disrupted in bladder urothelial carcinoma primarily related to "neurogenesis" and "cell differentiation" by integrated analysis of -omics data. Furthermore, we identified an intriguing collection of cancer-related genes that were deregulated at the levels of DNA methylation and mRNA expression, and we validated several of these genes (HIC1, SLIT2, RASAL1, and KRT17) by Bisulfite Sequencing PCR and Reverse Transcription qPCR in a panel of 33 bladder cancer samples.

Conclusions/significance: We characterized the profiles between methylome and transcriptome in bladder urothelial carcinoma, identified a set of significantly enriched key pathways, and screened four aberrantly methylated and expressed genes. Conclusively, our findings shed light on a new avenue for basic bladder cancer research.

Figure 1. Overview of DNA methylation patterns in relation to gene expression.

A. Genome wide DNA methylation, mRNA, and miRNA profiles from bladder cancer tissues and matched normal adjacent tissues. The log2-transformed average fold changes (tumor/normal) for methylation status (red), mRNA expression level (green), and miRNA expression level (blue) from all patients were drawn across the whole genome. The y-axis represents the log2 ratio, and the dashed lines represent the significance threshold (|log2Ratio| = 1). B. Distribution of the methylation levels in different types of genic regions between bladder cancer tissues and matched normal adjacent tissues. The box-plots for the distribution of DNA methylation levels based on the log2-transformed tag fold change (tumor/normal) from MMSDK across all patients were drawn. Types include CpG-rich promoters, CpG-poor promoters, exons, and introns.

Figure 2. Validation results of BSP for DNA methylation and RT-qPCR for gene expression.

A. Validation results of MMSDK with BSP. To compare the BSP-Sanger sequencing data and deep sequencing MMSDK data, the MluI loci that were determined to be differentially methylated on average by deep sequencing were validated using BSP. The height of the columns represents the log2-transformed average fold change (tumor/normal) in methylation level across the 9 patients. B. BSP and RT-qPCR results for four cancer-associated genes examined in 33 bladder cancer patients. The methylation levels of promoters of four selected genes (SLIT2, HIC1, RASRAl1, KRT17) and their expression level were evaluated in a panel of 33 samples. The height of the columns represents the log2 average fold change (tumor/normal) in methylation level (blue) or expression level (red) across all patients. The bars represent the standard error. The number of samples (n) used in the validation assay is indicated beside each standard error bar.