Yang Ziying, Wei Wen, Nie Daolin, Zhang Menglei, Chen Qiong
Department of Obstetrics and Gynecology, Gaoxin Branch of The First Affiliated Hospital of Nanchang University, Jiangxi, 330096 China.
Department of Obstetrics and Gynecology, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, 325027 China.
Cytotechnology. 2025 Aug;77(4):152. doi: 10.1007/s10616-025-00811-w. Epub 2025 Jul 18.
Ovarian cancer (OC) is the most lethal gynecologic malignancy, characterized by high recurrence rates and resistance to platinum-based chemotherapy. Epithelial-mesenchymal transition (EMT) is central to OC progression, where the transcription factor Snail plays a pivotal role in downregulating E-cadherin and promoting invasive, mesenchymal phenotypes. While multiple deubiquitinating enzymes (DUBs) have been implicated in stabilizing oncogenic proteins, the specific function of ubiquitin C-terminal hydrolase L3 (UCHL3) in OC remains unclear. This study investigates whether UCHL3 regulates Snail stability and thereby drives EMT and OC progression. Publicly available datasets (TCGA + GTEx) were analyzed to compare UCHL3 mRNA levels across various tumors and corresponding normal tissues. In vitro, UCHL3 expression was measured by qPCR and Western blot in an immortalized ovarian epithelial cell line (IOSE80) and four OC cell lines (SKOV3, ES2, OVCAR3, and A2780). Stable knockdowns of UCHL3 were generated using shRNA in SKOV3 and A2780 cells. Proliferation was evaluated by CCK-8 and colony formation assays, while invasion and migration capabilities were assessed using Matrigel invasion and Transwell migration assays. EMT marker expression was examined by qPCR and Western blot. Co-immunoprecipitation (Co-IP) determined the interaction between UCHL3 and Snail, and the effect of UCHL3 on Snail ubiquitination was explored using immunoprecipitation in the presence of MG132. A cycloheximide chase assay confirmed Snail protein stability. UCHL3 was significantly overexpressed in OC tissues compared to normal controls. Silencing UCHL3 in OC cells markedly impaired cell proliferation, migration, and invasion. Concomitantly, knockdown of UCHL3 reversed EMT features, evidenced by increased E-cadherin and decreased N-cadherin, Vimentin, and Snail protein levels. Co-IP experiments demonstrated that UCHL3 directly interacts with Snail, and loss of UCHL3 elevated Snail ubiquitination, leading to accelerated Snail protein degradation. These findings indicate that UCHL3 deubiquitinates and stabilizes Snail, promoting OC cell invasiveness and EMT. Our study identifies UCHL3 as a critical regulator of Snail-mediated EMT in OC. By stabilizing Snail, UCHL3 fosters malignancy-associated phenotypes, including enhanced proliferation, migration, and invasion. These results underscore the potential of targeting UCHL3 as a therapeutic strategy to disrupt Snail-driven EMT and impede OC progression.
The online version contains supplementary material available at 10.1007/s10616-025-00811-w.
卵巢癌（OC）是最致命的妇科恶性肿瘤，其特征是高复发率和对铂类化疗耐药。上皮-间质转化（EMT）是OC进展的核心，转录因子Snail在下调E-钙黏蛋白和促进侵袭性间质表型中起关键作用。虽然多种去泛素化酶（DUBs）与稳定致癌蛋白有关，但泛素C末端水解酶L3（UCHL3）在OC中的具体功能仍不清楚。本研究调查UCHL3是否调节Snail稳定性，从而驱动EMT和OC进展。分析公开可用数据集（TCGA + GTEx）以比较UCHL3 mRNA在各种肿瘤和相应正常组织中的水平。在体外，通过qPCR和蛋白质印迹法在永生化卵巢上皮细胞系（IOSE80）和四种OC细胞系（SKOV3、ES2、OVCAR3和A2780）中测量UCHL3表达。使用shRNA在SKOV3和A2780细胞中稳定敲低UCHL3。通过CCK-8和集落形成试验评估增殖，同时使用基质胶侵袭和Transwell迁移试验评估侵袭和迁移能力。通过qPCR和蛋白质印迹法检测EMT标志物表达。免疫共沉淀（Co-IP）确定UCHL3与Snail之间的相互作用，并在存在MG132的情况下使用免疫沉淀法探索UCHL3对Snail泛素化的影响。环己酰亚胺追踪试验证实了Snail蛋白的稳定性。与正常对照相比，UCHL3在OC组织中显著过表达。在OC细胞中沉默UCHL3明显损害细胞增殖、迁移和侵袭。同时，敲低UCHL3逆转了EMT特征，表现为E-钙黏蛋白增加，N-钙黏蛋白、波形蛋白和Snail蛋白水平降低。Co-IP实验表明UCHL3直接与Snail相互作用，UCHL3的缺失增加了Snail的泛素化，导致Snail蛋白降解加速。这些发现表明UCHL3去泛素化并稳定Snail，促进OC细胞侵袭和EMT。我们的研究确定UCHL3是OC中Snail介导的EMT的关键调节因子。通过稳定Snail，UCHL3促进与恶性肿瘤相关的表型，包括增强的增殖、迁移和侵袭。这些结果强调了靶向UCHL3作为一种治疗策略来破坏Snail驱动的EMT并阻碍OC进展的潜力。
在线版本包含可在10.1007/s10616-025-00811-w获取的补充材料。
