METHODS: Selenium-binding protein 1 (SELENBP1), USP15, CCL2/5, CXCL10/11, IL-4, and TGF-β1 mRNA levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR). SELENBP1, USP15, GPX4, IL-10, Arg-1, Granzyme B, TNF-α, and PR domain zinc finger protein 1 (PRDM1) protein levels were examined by western blot assay. Fe+ level, malondialdehyde (MDA), and lipid-ROS levels were determined using special kits. The proportion of CD11b+CD206+ positive cells was detected using a flow cytometry assay. The role of SELENBP1 on TC cell growth was examined using a xenograft tumor model in vivo. After GeneMANIA prediction, the interaction between USP15 and SELENBP1 was verified using Co-immunoprecipitation (CoIP) assay. The binding between PRDM1 and USP15 promoter was predicted by JASPAR and validated using Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays.
RESULTS: SELENBP1 was increased in TC subjects and cell lines, and its knockdown repressed TC cell proliferation, migration, invasion, immune escape, and induced ferroptosis in vitro, as well as blocked tumor growth in vivo. In mechanism, USP15 interacted with SELENBP1 and maintained its stabilization by removing ubiquitin. Meanwhile, the upregulation of USP15 was induced by the transcription factor PRDM1.
CONCLUSIONS: USP15 transcriptionally mediated by PRDM1 might boost TC cell malignant behaviors through deubiquitinating SELENBP1, providing a promising therapeutic target for TC treatment.
方法:硒结合蛋白1(SELENBP1),使用实时定量聚合酶链反应(RT-qPCR)检测USP15,CCL2/5,CXCL10/11,IL-4和TGF-β1mRNA水平。SELENBP1,USP15,GPX4,IL-10,Arg-1,粒酶B,TNF-α,和PR结构域锌指蛋白1(PRDM1)蛋白水平通过蛋白质印迹法检测。Fe+水平,丙二醛(MDA),使用特殊试剂盒测定脂质-ROS水平。使用流式细胞术测定检测CD11b+CD206+阳性细胞的比例。在体内使用异种移植肿瘤模型检查SELENBP1对TC细胞生长的作用。遗传MANIA预测后,USP15和SELENBP1之间的相互作用使用免疫共沉淀(CoIP)分析进行了验证.PRDM1和USP15启动子之间的结合由JASPAR预测,并使用染色质免疫沉淀(ChIP)和双荧光素酶报告基因测定进行验证。
结果:在TC受试者和细胞系中SELENBP1增加,它的敲除抑制了TC细胞的增殖,迁移,入侵,免疫逃逸,并在体外诱导铁凋亡,以及在体内阻断肿瘤生长。在机制上,USP15与SELENBP1相互作用并通过去除泛素维持其稳定性。同时,转录因子PRDM1诱导了USP15的上调。
结论:由PRDM1转录介导的USP15可能通过去泛素化SELENBP1促进TC细胞的恶性行为,为TC治疗提供了一个有希望的治疗靶点。