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

Hsiang-Ying Lee; Ching-Chia Li; Wei-Ming Li; Ya-Ling Hsu; Hsin-Chih Yeh; Hung-Lung Ke; Bi Wen Yeh; Chun-Nung Huang; Chien-Feng Li; Po-Lin Kuo; Wen-Jeng Wu

doi:10.7717/peerj.11343

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

PeerJ. 2021 Apr 27:9:e11343. doi: 10.7717/peerj.11343. eCollection 2021.

Authors

Hsiang-Ying Lee^{1

2

3

4}, Ching-Chia Li^{3

4

5}, Wei-Ming Li^{3

4

5

6}, Ya-Ling Hsu⁵, Hsin-Chih Yeh^{2

3

4

5}, Hung-Lung Ke^{3

4

5}, Bi Wen Yeh^{3

4}, Chun-Nung Huang^{2

3

4

5}, Chien-Feng Li⁷, Po-Lin Kuo^{1

8

9}, Wen-Jeng Wu^{3

4

5

9}

Affiliations

¹ Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
² Department of Urology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan.
³ Department of Urology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
⁴ Department of Urology, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁵ Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁶ Department of Urology, Ministry of Health and Welfare Pingtung Hospital, Pingtung, Taiwan.
⁷ Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan.
⁸ Center for Biomarkers and Biotech Drugs, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁹ Institute of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung, Taiwan.

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.

Keywords: Bioinformatics; Next-generation sequencing; PDE5A; Target genes; Upper tract urothelial carcinoma.

Grants and funding

This study was supported by grants from the Ministry of Science and Technology (MOST 106-2314-B-037-029), the Kaohsiung Medical University Hospital (KMUH 106-6R53), Kaohsiung Municipal Ta-Tung Hospital (kmtth-108-R003) and partially by the Kaohsiung Medical University Grant (KMU-KI109002), the Regenerative Medicine and Cell Therapy Research Center Grant (KMU-TC108A02), and the Cohort Research Center (KMU-TC108B07). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.