The catenin beta 1 gene (CTNNB1) plays a crucial role in the malignant progression of various cancers. Recent studies have suggested that CTNNB1 hyperactivation is closely related to the occurrence and development of bladder cancer (BCa). As a member of the deubiquitinating enzyme (DUB) family, ubiquitin C-terminal hydrolase L3 (UCHL3) is abnormally expressed in various cancers. In this study, we discovered that UCHL3 is a novel oncogene in bladder cancer, suggesting it is a promising target against bladder cancer. 我们利用CRISPR-Cas9技术构建了稳定过表达或敲除UCHL3的细胞系。利用Western blotting确定了UCHL3的成功过表达或敲除。然后，我们进行了CCK-8、克隆形成、软琼脂和Transwell迁移实验，以确定UCHL3基因对细胞表型的影响。我们使用UCHL3缺陷的T24细胞（通过CRISPR-Cas9介导的基因组编辑建立）进行了RNA-seq。我们通过热图和基因集富集分析（GSEA）分析了野生型和UCHL3缺陷的T24细胞系中WNT信号通路基因表达的差异。然后，我们使用双荧光报告基因检测验证了UCHL3对Wnt信号通路的影响。接下来，我们使用Western blotting、共免疫沉淀和免疫荧光实验分析了相关机制。我们还进行了裸鼠肿瘤形成实验。此外，我们建立了条件性UCHL3敲除小鼠和膀胱癌模型小鼠以进行研究。 我们发现UCHL3的过表达促进了膀胱癌细胞的增殖、侵袭和迁移，而膀胱癌细胞中UCHL3的减少延缓了体外和体内的肿瘤发生。UCHL3与Wnt信号通路密切相关并引发了Wnt信号通路的激活，表明其功能取决于其去泛素化活性。值得注意的是，UCHL3缺陷小鼠对膀胱肿瘤发生的敏感性较低。此外，UCHL3在膀胱癌细胞中高度表达，与晚期病理指标相关。 总之，我们发现UCHL3在膀胱癌中存在扩增，并在体外增强肿瘤细胞的增殖和迁移，在体内促进膀胱肿瘤的发生和发展。此外，我们揭示了UCHL3稳定CTNNB1表达，进而激活致癌性的Wnt信号通路。因此，我们的研究结果强烈提示UCHL3是膀胱癌的有希望的治疗靶点。© 2023. BioMed Central有限公司，属于Springer Nature的一部分。

The catenin beta 1 gene (CTNNB1) plays a crucial role in the malignant progression of various cancers. Recent studies have suggested that CTNNB1 hyperactivation is closely related to the occurrence and development of bladder cancer (BCa). As a member of the deubiquitinating enzyme (DUB) family, ubiquitin C-terminal hydrolase L3 (UCHL3) is abnormally expressed in various cancers. In this study, we discovered that UCHL3 is a novel oncogene in bladder cancer, suggesting it is a promising target against bladder cancer.We utilized CRISPR‒Cas9 technology to construct cell lines with UCHL3 stably overexpressed or knocked out. The successful overexpression or knockout of UCHL3 was determined using Western blotting. Then, we performed CCK-8, colony formation, soft agar and Transwell migration assays to determine the impact of the UCHL3 gene on cell phenotype. RNA-seq was performed with UCHL3-depleted T24 cells (established via CRISPR-Cas9-mediated genomic editing). We analyzed differences in WNT pathway gene expression in wild-type and UCHL3-deficient T24 cell lines using a heatmap and by gene set enrichment analysis (GSEA). Then, we validated the effect of UCHL3 on the Wnt pathway using a dual fluorescence reporter. We then analyzed the underlying mechanisms involved using Western blots, co-IP, and immunofluorescence results. We also conducted nude mouse tumor formation experiments. Moreover, conditional UCHL3-knockout mice and bladder cancer model mice were established for research.We found that the overexpression of UCHL3 boosted bladder cancer cell proliferation, invasion and migration, while the depletion of UCHL3 in bladder cancer cells delayed tumor tumorigenesis in vitro and in vivo. UCHL3 was highly associated with the Wnt signaling pathway and triggered the activation of the Wnt signaling pathway, which showed that its functions depend on its deubiquitination activity. Notably, Uchl3-deficient mice were less susceptible to bladder tumorigenesis. Additionally, UCHL3 was highly expressed in bladder cancer cells and associated with indicators of advanced clinicopathology.In summary, we found that UCHL3 is amplified in bladder cancer and functions as a tumor promoter that enhances proliferation and migration of tumor cells in vitro and bladder tumorigenesis and progression in vivo. Furthermore, we revealed that UCHL3 stabilizes CTNNB1 expression, resulting in the activation of the oncogenic Wnt signaling pathway. Therefore, our findings strongly suggest that UCHL3 is a promising therapeutic target for bladder cancer.© 2023. BioMed Central Ltd., part of Springer Nature.