Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, 430071, Hubei, China.
Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, 430072, Hubei, China.
J Transl Med. 2023 Sep 22;21(1):656. doi: 10.1186/s12967-023-04311-3.
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.
连接蛋白β 1 基因（CTNNB1）在各种癌症的恶性进展中起着至关重要的作用。最近的研究表明，CTNNB1 过度激活与膀胱癌（BCa）的发生和发展密切相关。泛素 C 端水解酶 L3（UCHL3）作为去泛素化酶（DUB）家族的一员，在各种癌症中异常表达。在这项研究中，我们发现 UCHL3 是膀胱癌的一种新型癌基因，表明它是治疗膀胱癌的有前途的靶点。
我们利用 CRISPR-Cas9 技术构建了 UCHL3 稳定过表达或敲除的细胞系。Western blot 检测 UCHL3 的过表达或敲除是否成功。然后，我们进行了 CCK-8、集落形成、软琼脂和 Transwell 迁移实验，以确定 UCHL3 基因对细胞表型的影响。用 UCHL3 耗尽的 T24 细胞（通过 CRISPR-Cas9 介导的基因组编辑建立）进行 RNA-seq。我们使用热图和基因集富集分析（GSEA）分析野生型和 UCHL3 缺陷型 T24 细胞系中 WNT 通路基因表达的差异。然后，我们使用双荧光报告验证 UCHL3 对 Wnt 通路的影响。然后，我们使用 Western blot、co-IP 和免疫荧光结果分析了涉及的潜在机制。我们还进行了裸鼠肿瘤形成实验。此外，还建立了条件性 UCHL3 敲除小鼠和膀胱癌模型小鼠进行研究。
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 的耗竭则延缓了体外和体内肿瘤的发生。UCHL3 与 Wnt 信号通路高度相关，并触发了 Wnt 信号通路的激活，表明其功能依赖于其去泛素化活性。值得注意的是，Uchl3 缺陷型小鼠对膀胱癌的易感性较低。此外，UCHL3 在膀胱癌细胞中高表达，并与高级临床病理指标相关。
综上所述，我们发现 UCHL3 在膀胱癌中扩增，并作为一种肿瘤促进剂发挥作用，增强肿瘤细胞在体外的增殖和迁移以及体内膀胱癌的发生和进展。此外，我们揭示了 UCHL3 稳定 CTNNB1 的表达，导致致癌 Wnt 信号通路的激活。因此，我们的研究结果强烈表明 UCHL3 是膀胱癌有前途的治疗靶点。
