USP5 promotes epithelial-mesenchymal transition by stabilizing SLUG in hepatocellular carcinoma.,Theranostics

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USP5 promotes epithelial-mesenchymal transition by stabilizing SLUG in hepatocellular carcinoma.
Theranostics ( IF 12.4 ) Pub Date : 2019-01-01 , DOI: 10.7150/thno.27654
Jing Meng ₁ , Xiaoyu Ai ₁ , Yueyang Lei ₁ , Weilong Zhong ₁ , Baoxin Qian ₂ , Kailiang Qiao ₁ , Xiaorui Wang ₃ , Bijiao Zhou ₁ , Hongzhi Wang ₁ , Longcong Huai ₁ , Xiaoyun Zhang ₁ , Jingxia Han ₄ , Yinyin Xue ₁ , Yuan Liang ₁ , Honggang Zhou _{1,

4} , Shuang Chen ₄ , Tao Sun _{1,

4} , Cheng Yang _{1,

4}

Affiliation

Rationale: The role of SLUG in epithelial-mesenchymal transition during tumor progression has been thoroughly studied, but its precise regulation remains poorly explored. Methods: The affinity purification, mass spectrometry and CO-IP were performed to identify the interaction between SLUG and ubiquitin-specific protease 5 (USP5). Cycloheximide chase assays and deubiquitination assays confirmed that the effect of USP5 on the deubiquitin of SLUG. The dual-luciferase reporter and chromatin immunoprecipitation assays were employed to observe the direct transcriptional regulation of E-cadherin by SLUG effected by USP5. EMT related markers was detected by western blotting and immunofluorescence. Molecular docking, SPR sensor (biacore) and co-location were detected to prove Formononetin targets USP5. Bioinformatics analysis was used to study the relation of USP5 and SLUG to malignancy degree of HCC. Cell migration, invasion in HCC cells and xenografts model in nude mouse were conducted to detect the promotion of USP5 and the inhibition of Formononetin on EMT. Results: USP5 interacts with and stabilizes SLUG to regulate its abundance through USP5 deubiquitination activities in epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC). USP5 is highly expressed and positively correlated with SLUG expression in HCC with high malignancy. Knockdown of USP5 inhibits SLUG deubiquitination and inhibits HCC cells proliferation, metastasis, and invasion, while overexpression of USP5 promotes SLUG stability and EMT in vitro and in vivo. Through virtual screening, we found that Formononetin exhibits excellent binding to USP5. Moreover, Formononetin inhibits deubiquitinating activities of USP5 to SLUG and consequently impedes the EMT and malignant progression of HCC. Conclusion: Our findings reveal that USP5 serve as a potential target for tumor intervention and provide a preliminary antitumor therapy for inhibit EMT by targeting USP5 or its interaction with SLUG in HCC.

中文翻译：

USP5 通过稳定肝细胞癌中的 SLUG 来促进上皮间质转化。

理由：SLUG 在肿瘤进展过程中上皮-间质转化中的作用已得到深入研究，但其精确调控仍知之甚少。方法：通过亲和纯化、质谱和CO-IP 鉴定SLUG 与泛素特异性蛋白酶5 (USP5) 之间的相互作用。放线菌酮追踪实验和去泛素化实验证实了USP5对SLUG去泛素化的影响。采用双荧光素酶报告基因和染色质免疫沉淀实验观察USP5对SLUG对E-cadherin的直接转录调控。通过蛋白质印迹和免疫荧光检测EMT相关标志物。检测到分子对接、SPR 传感器 (biacore) 和共定位，证明芒柄花素靶向 USP5。采用生物信息学分析研究USP5和SLUG与HCC恶性程度的关系。通过肝癌细胞的细胞迁移、侵袭以及裸鼠异种移植模型检测USP5对EMT的促进作用和芒柄花素对EMT的抑制作用。结果：USP5 在肝细胞癌 (HCC) 的上皮间质转化 (EMT) 中通过 USP5 去泛素化活性与 SLUG 相互作用并稳定 SLUG，从而调节其丰度。USP5在高度恶性的HCC中高表达且与SLUG表达呈正相关。USP5的敲除抑制SLUG去泛素化并抑制HCC细胞的增殖、转移和侵袭，而USP5的过表达则促进SLUG在体外和体内的稳定性和EMT。通过虚拟筛选，我们发现芒柄花素与 USP5 具有良好的结合力。此外，芒柄花素可抑制 USP5 对 SLUG 的去泛素化活性，从而阻止 HCC 的 EMT 和恶性进展。结论：我们的研究结果表明，USP5作为肿瘤干预的潜在靶点，并为HCC中通过靶向USP5或其与SLUG的相互作用抑制EMT提供了初步的抗肿瘤治疗。

更新日期：2019-01-01

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