Additional file 1: of Identification of epigenetic interactions between miRNA and DNA methylation associated with gene expression as potential prognostic markers in bladder cancer

Supplementary information. Table S1. Significant epigenetic interactions between miRNA and methylation associated with target genes. Table S2. Significant epigenetic interactions between miRNA and methylation associated with target genes for papillary subtype. Table S3. Significant epigenetic interactions between miRNA and methylation associated with target genes for non-papillary subtype. Table S4. Summary of overall survival analysis results. Figure S1. Venn Diagram of Significant target genes for papillary, non-papillary subtypes. Figure S2. Survival analysis between two subgroups (LL and HH). Figure S3. Gene expression boxplot for two subgroups (LL and HH). Figure S4. Gene expression boxplot for four subgroups (LL, LH, HL, and HH). Figure S5. Survival analysis across four subgroups (LL, LH, HL, and HH). (DOCX 2368 kb

The effects of alternative splicing on miRNA binding sites in bladder cancer

<div><p>Eukaryotic organisms have developed a variety of mechanisms to regulate translation post-transcriptionally, including but not limited to the use of miRNA silencing in many species. One method of post-transcriptional regulation is through miRNAs that bind to the 3′ UTRs to regulate mRNA abundance and influence protein expression. Therefore, the diversity of mRNA 3′ UTRs mediating miRNA binding sites influence miRNA-mediated regulation. Alternative polyadenylation, by shortening mRNA isoforms, increases the diversity of 3′ UTRs; moreover, short mRNA isoforms elude miRNA-medicated repression. Because no current prediction methods for putative miRNA target sites consider whether or not 1) splicing-informed miRNA binding sites and/or 2) the use of 3′ UTRs provide higher resolution or functionality, we sought to identify not only the genome-wide impact of using exons in mRNA 3′ UTRs but also their functional connection to miRNA regulation and clinical outcomes in cancer. With a genome-wide expression of mRNA and miRNA quantified by 395 bladder cancer cases from The Cancer Genome Atlas (TCGA), we 1) demonstrate the diversity of 3′ UTRs affecting miRNA efficiency and 2) identify a set of genes clinically associated with mRNA expression in bladder cancer. Knowledge of 3′ UTR diversity will not only be a useful addition to current miRNA target prediction algorithms but also enhance the clinical utility of mRNA isoforms in the expression of mRNA in cancer. Thus, variability among cancer patient’s variability in molecular signatures based on these exon usage events in 3′ UTR along with miRNAs in bladder cancer may lead to better prognostic/treatment strategies for improved precision medicine.</p></div

Case study of the <i>BACE1</i> gene, which shows statistically significant differential expression of miRNA-mediated transcript isoforms between stage status and histology status—but not survival outcome.

<p>For plots A and B, the x-axes represent miRNA expression, and the y-axes represent the relative ratio of miRNA-mediated transcript isoforms expression to overall transcript isoforms expression per single miRNA (i.e., hsa-miR-17-5p). The p-value was corrected with the Bonferroni method. (A) Red dots and blue dots represent stage 0 and stage 1 of bladder cancer, respectively. (B) Red dots and blue dots represent histology 0 and histology 1 of bladder cancer, respectively. (C) In the two groups of bladder cancer cases that expressed high PSI and low PSI, we observed no differences in survival outcome.</p

Study design overview.

<p>Step 1 involved constructing a comprehensive set of relationships between mRNA and miRNA by compiling three existing miRNA target databases: miTarBase, TargetScan, and miRanda. Step 2 involved searching for the miRNA-binding exons (MBEs) and identifying which transcript isoforms retain or do not retain MBEs. When a transcript isoform loses miRNA binding sites in the 3′ UTR due to one of these events—i.e., 1) exon skipping (miRNA 2 in mRNA2), 2) alternative splice 3′ or 5′ splice sites (miRNA 3 in mRNA1), 3) mutually exclusive 3′ UTR regions (i.e. mRNA3 vs. mRNA5 and mRNA5 vs. mRNA6), defining the case that miRNA binding sites in the genomic regions translated into the two mRNAs do not overlap each other at all, and 4) others in these three cases (i.e., retained introns, non-coding RNA, and alternative polyadenylation)—it was assigned to miRNA-binding Group A; otherwise, it was assigned to Group B. Steps 3, 4, and 5 were to not only identify alternative splicing isoforms and splicing events, but also calculate FPKM as a quantitative expression level using TopHat and Cufflinks. We used level 3 data for miRNA expression in the TCGA. Step 6 integrated comprehensive sets of MBEs status in 3′ UTRs with the expression of miRNA and mRNA and, therefore, estimated the relative expression ratio between Group A (i.e., transcript isoforms repressed by miRNA, defined by MBE-retaining mRNA) and all mRNA expression. This is a normalized measurement of the miRNA-mediated repression ratio to the overall transcript expressions per single gene. The multiplicity was corrected by the Bonferroni method. Step 7 first tested an association of differential expression of miRNA-mediated transcript isoforms in the two-stage and histology groups and then predicted overall survival time in the bladder cancer cases.</p

Case study of the <i>VEGFA</i> gene, which shows statistically significant differential expression of miRNA-mediated transcript isoforms between stage status, histology status, and survival outcomes.

<p>For plots A and B, the x-axes represent miRNA expression, and the y-axes represent the relative ratio of miRNA-mediated transcript isoforms expression to overall transcript isoforms expression per a single miRNA (i.e., hsa-miR-361-5p). The p-value was corrected with the Bonferroni method (A) Red dots and blue dots represent stage 0 and stage 1 of bladder cancer, respectively. (B) Red dots and blue dots represent histology 0 and histology 1 of bladder cancer, respectively. (C) In the two groups of bladder cancer cases that expressed high PSI and low PSI, the later showed better survival outcomes, compared to the latter. As expected, more severe stage group 0 (i.e., purple plot in (D) and histology group 1 (i.e., green plot in E) exhibited a shorter expected survival time.</p