Identification of potential genes in upper tract urothelial carcinoma using next-generation sequencing with bioinformatics and in vitro analyses

Abstract

Background 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). In Taiwan, this figure is markedly higher, where it can reach up to 30% of UC cases. Materials and Methods 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® 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. Results Interactions 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. Conclusions Our 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.

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“…We observed PDE5A overexpression in PTC tissues, which is consistent with earlier observations. However, PDE5A is downregulated in other tumor types like upper tract urothelial carcinoma [27] and melanoma [28]. Interestingly, Nagel et al reported that PDE5A has an anti-apoptotic function by reducing the level of cGMP in the cytoplasm, suggesting that PDE5A is an oncogene in Hodgkin's lymphoma [29].…”

Current Development and Application of Anaerobic Glycolytic Enzymes in Urothelial Cancer

“…In this study, they found that some tyrosine kinases may be potential treatment strategies. Lee et al and Chen et al used an RNA sequencing approach to distinguish normal/tumor pairs in patients with urothelial cancer (GSE159824 and GSE133624) [ 122 , 123 ]. Many scientists have generated their data from patient-derived xenografts (PDXs) and organoids (GSE155007 and GSE134292) [ 124 ].…”