Abstract
BackgroundWe aimed to identify prognostic biomarkers of upper tract urothelial carcinomas (UTUCs), including microRNAs (miRNAs) and genes which account for only 5% to 10% of all urothelial carcinomas (UCs). In Taiwan, this figure is markedly higher, where it can reach up to 30% of UC cases.Materials and MethodsUsing next-generation sequencing (NGS), we analyzed two pairs of renal pelvis tumors and adjacent normal urothelial tissues to screen miRNAs and messenger RNAs. By combining bioinformatics analysis from miRmap, Gene Expression Omnibus (GEO), and Oncomine and Ingenuity® Pathway Analysis databases, we identified candidate genes. To search for upstream miRNAs with exact target binding sites, we used miRmap, TargetScan, and miRDB to enforce evidence. Then, we clarified gene and protein expression through an in vitro study using western blot analysis and quantitative real-time reverse transcriptase-PCR.ResultsInteractions between selected target genes obtained using the NGS and miRmap methods were assessed through a Venn diagram analysis. Six potential genes, namely, PDE5A, RECK, ZEB2, NCALD, PLCXD3 and CYBRD1 showed significant differences. Further analysis of gene expression from the GEO dataset indicated lower expression of PDE5A, RECK, ZEB2, and CYBRD1 in bladder cancer tissue than in normal bladder mucosa, which indicated that PDE5A, RECK, ZEB2, and CYBRD1 may act as tumor suppressors in UTUC. In addition, we compared the expression of these genes in various UC cell lines (RT4, BFTC905, J82, T24, UMUC3, 5637, BFTC 909, UMUC14) and found decreased expression of PDE5A in muscle-invasive UC cells compared with the RT4 cell line. Furthermore, by using paired UTUC and normal tissues from 20 patients, lower PDE5A expression was also demonstrated in tumor specimens.ConclusionsOur findings suggest these candidate genes may play some roles in UTUC progression. We propose that these markers may be potential targets clarified by in vitro and in vivo experiments. PDE5A also potentially presents tumor suppressor genes, as identified by comparing the expression between normal and tumor specimens.
Highlights
Urothelial carcinomas (UCs) are derived from the urothelium and divided into upper and lower tract urothelial carcinomas (UCs) according to tumor location
The purpose of this study was to discover valuable biomarkers using a combination of bioinformatics tools including miRmap (Vejnar, Blum & Zdobnov, 2013), Gene Expression Omnibus (GEO), Oncomine (Rhodes et al, 2007), Ingenuity R Pathway Analysis (IPA) (Zhao, Liu & Qu, 2017), TargetScan, and miRDB (Wong & Wang, 2015) in our upper tract urothelial carcinomas (UTUCs) patient specimens focused on renal pelvis cancer
The overlapping genes between miRNA putative targets and differentially expressed genes of our dataset were identified by Venn diagram analysis, and six potential genes, namely, Phosphodiesterase 5A (PDE5A), RECK, ZEB2, NCALD, PLCXD3, and Cytochrome b reductase 1 (CYBRD1) had significant differences (Fig. 2)
Summary
Urothelial carcinomas (UCs) are derived from the urothelium and divided into upper and lower tract UCs according to tumor location. Upper tract urothelial carcinoma (UTUC), originating from the renal pelvis and ureter presents unique features in Taiwan compared to those in the Western regions. We aimed to identify prognostic biomarkers of upper tract urothelial carcinomas (UTUCs), including microRNAs (miRNAs) and genes which account for only 5% to 10% of all urothelial carcinomas (UCs). Using next-generation sequencing (NGS), we analyzed two pairs of renal pelvis tumors and adjacent normal urothelial tissues to screen miRNAs and messenger RNAs. By combining bioinformatics analysis from miRmap, Gene Expression Omnibus (GEO), and Oncomine and Ingenuity R Pathway Analysis databases, we identified candidate genes. Further analysis of gene expression from the GEO dataset indicated lower expression of PDE5A, RECK, ZEB2, and CYBRD1 in bladder cancer tissue than in normal bladder mucosa, which indicated that PDE5A, RECK, ZEB2, and CYBRD1 may act as tumor suppressors in UTUC.
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