Lee Hsiang-Ying, Li Ching-Chia, Li Wei-Ming, Hsu Ya-Ling, Yeh Hsin-Chih, Ke Hung-Lung, Yeh Bi Wen, Huang Chun-Nung, Li Chien-Feng, Kuo Po-Lin, Wu Wen-Jeng
Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Department of Urology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan.
PeerJ. 2021 Apr 27;9:e11343. doi: 10.7717/peerj.11343. eCollection 2021.
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.
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.
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.
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.
我们旨在鉴定上尿路尿路上皮癌（UTUC）的预后生物标志物，包括仅占所有尿路上皮癌（UC）5%至10%的微小RNA（miRNA）和基因。在台湾，这一比例明显更高，可达UC病例的30%。
我们使用下一代测序（NGS）分析了两对肾盂肿瘤及相邻正常尿路上皮组织，以筛选miRNA和信使RNA。通过整合来自miRmap、基因表达综合数据库（GEO）、Oncomine和 Ingenuity通路分析数据库的生物信息学分析，我们鉴定出候选基因。为了寻找具有精确靶标结合位点的上游miRNA，我们使用miRmap、TargetScan和miRDB来强化证据。然后，我们通过蛋白质印迹分析和定量实时逆转录聚合酶链反应的体外研究来阐明基因和蛋白质表达。
通过维恩图分析评估了使用NGS和miRmap方法获得的选定靶基因之间的相互作用。六个潜在基因，即磷酸二酯酶5A（PDE5A）、富含半胱氨酸的分泌蛋白（RECK）、锌指E盒结合蛋白2（ZEB2）、神经元钙结合蛋白（NCALD）、磷脂酶C类结构域包含蛋白3（PLCXD3）和细胞色素b还原酶1（CYBRD1）表现出显著差异。对GEO数据集基因表达的进一步分析表明，膀胱癌组织中PDE5A、RECK、ZEB2和CYBRD1的表达低于正常膀胱黏膜，这表明PDE5A、RECK、ZEB2和CYBRD1可能在UTUC中起肿瘤抑制作用。此外，我们比较了这些基因在各种UC细胞系（RT4、BFTC905、J82、T24、UMUC3、5637、BFTC 909、UMUC14）中的表达，发现与RT4细胞系相比，肌肉浸润性UC细胞中PDE5A的表达降低。此外，通过使用来自20名患者的配对UTUC和正常组织，肿瘤标本中也显示出较低的PDE5A表达。
我们的研究结果表明，这些候选基因可能在UTUC进展中发挥一定作用。我们提出，这些标志物可能是通过体外和体内实验阐明的潜在靶点。通过比较正常和肿瘤标本之间的表达，PDE5A也可能是潜在的肿瘤抑制基因。
