Zhang Yingzi, Lu Yue, Xu Yiqing, Le Ziyu, Liu Yi, Tu Wenzhi, Liu Yong
Department of Radiation Oncology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201620, China.
Department of Radiotherapy, Huangpu Branch of the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.
Life Sci. 2023 May 15;321:121594. doi: 10.1016/j.lfs.2023.121594. Epub 2023 Mar 18.
Hypoxia is an important feature of nasopharyngeal carcinoma (NPC). "Protein interacting with PRKCA 1" (PICK1) is commonly downregulated in human malignancies and is functionally related to poor prognosis. However, there is a limited understanding of the upstream mechanisms regulating PICK1 currently.
PICK1 and HIF-1α expression levels were analyzed by Immunohistochemistry (IHC), western blotting, and quantitative real-time PCR assay. Protein stability and ubiquitin assays were used to investigate PICK1 protein degradation. Immunofluorescence and co-immunoprecipitation assays were used to demonstrate the interaction between RBCK1 and PICK1. Gene knockdown by siRNA transfection was used to investigate the role of HIF-1α and RBCK1 in hypoxia-induced PICK1 degradation. Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) assays and subcutaneous xenograft nude models were used to explore the roles of RBCK1 and PICK1 in NPC cell proliferation.
PICK1 expression in NPC tissue was negatively relative to that of HIF-1α. HIF-1α downregulated PICK1 expression by facilitating its ubiquitination by the E3 ligases RANBP2-type and C3HC4-type zinc finger containing 1 (RBCK1), thereby enhancing proteasome-mediated PICK1 degradation. RBCK1 knockdown inhibited NPC cell proliferation, which was ameliorated by double knockdown of RBCK1/PICK1.
These data provide evidence for an NPC cell adaptation mechanism to hypoxia, where HIF-1α regulates RBCK1, which targets PICK1 for degradation to promote cell proliferation.
缺氧是鼻咽癌（NPC）的一个重要特征。“与PRKCA 1相互作用的蛋白质”（PICK1）在人类恶性肿瘤中通常下调，并且在功能上与预后不良相关。然而，目前对调节PICK1的上游机制了解有限。
通过免疫组织化学（IHC）、蛋白质印迹法和定量实时PCR分析PICK1和HIF-1α的表达水平。使用蛋白质稳定性和泛素分析来研究PICK1蛋白降解。免疫荧光和免疫共沉淀分析用于证明RBCK1与PICK1之间的相互作用。通过小干扰RNA（siRNA）转染进行基因敲低，以研究HIF-1α和RBCK1在缺氧诱导的PICK1降解中的作用。使用细胞计数试剂盒-8（CCK-8）、5-乙炔基-2'-脱氧尿苷（EdU）分析和皮下异种移植裸鼠模型来探讨RBCK1和PICK1在NPC细胞增殖中的作用。
NPC组织中PICK1的表达与HIF-1α的表达呈负相关。HIF-1α通过促进E3连接酶含RANBP2型和C3HC4型锌指蛋白1（RBCK1）对PICK1的泛素化，下调PICK1的表达，从而增强蛋白酶体介导的PICK1降解。RBCK1敲低抑制NPC细胞增殖，而RBCK1/PICK1双敲低可改善这种抑制作用。
这些数据为NPC细胞对缺氧的适应机制提供了证据，即HIF-1α调节RBCK1，RBCK1靶向PICK1进行降解以促进细胞增殖。
