神经胶质瘤U251细胞中TRIM21和TRIM8之间相互作用研究  

Study of Mutual Regulation between TRIM21 and TRIM8 in Glioma U251 Cells

作　　者：王琳 刘雪梅 李慧 黄皑雪 赵越超 肖参 高波[1] 邵宁生 WANG Lin;LIU Xuemei;LI Hui;HUANG Aixue;ZHAO Yuechao;XIAO Can;GAO Bo;SHAO Ningsheng(Institute of Military Cognition and Brain Sciences,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing,100850,China)

机构地区：[1]军事科学院军事医学研究院军事认知与脑科学研究所,北京市100850

出　　处：《医学分子生物学杂志》2024年第3期187-195,共9页Journal of Medical Molecular Biology

基　　金：国家自然科学基金(No.3120566,No.31370794)。

摘　　要：目的探讨TRIM21和TRIM8在神经胶质瘤U251细胞中的相互作用机制及其生物学效应。方法在U251细胞中分别过表达和敲低TRIM21或TRIM8,通过蛋白质印迹和RT-PCR法检测两者的蛋白和mRNA表达水平。利用免疫荧光实验分析TRIM21和TRIM8的亚细胞定位。通过免疫共沉淀实验验证TRIM21和TRIM8之间的相互作用。应用胞内泛素化实验检测TRIM21和TRIM8的泛素化修饰。蛋白酶体抑制剂MG-132用于抑制蛋白酶体活性。利用流式细胞术检测U251细胞凋亡。结果在U251细胞中,TRIM21负向调控TRIM8蛋白水平,反之,TRIM8也可以负向调控TRIM21蛋白水平,并且都能抑制细胞凋亡。机制研究显示,U251细胞中TRIM21和TRIM8之间存在相互结合,两者相互调控是通过催化泛素化介导的泛素-蛋白酶体依赖性降解途径实现的。结论U251细胞中TRIM21与TRIM8可以通过泛素化修饰促进泛素-蛋白酶体依赖性的蛋白质降解相互负向调控,提示体内细胞正常生长分化需要TRIM21-TRIM8之间蛋白质水平的相对稳态,也即可能存在E3泛素连接酶蛋白稳态,稳态失衡可能与肿瘤的发生发展密切相关。Objective To explore the mutual regulation between TRIM21 and TRIM8 in glioma U251 cells.Methods TRIM21 and TRIM8 were overexpressed or knocked down respectively in U251 cells,and their protein and mRNA expression levels were detected by Western blotting and RT-PCR respectively.Immunofluorescence experiment was used to analyze the subcellular localization of TRIM21 and TRIM8.The interaction between TRIM21 and TRIM8 was verified by immunoprecipitation experiment.The ubiquitination modification of TRIM21 or TRIM8 was analyzed by intracellular ubiquitination experiment.Protease inhibitor MG-132 was used to inhibit protease activity.U251 cell apoptosis was detected by flow cytometry assay.Results TRIM21 and TRIM8 could negatively regulate each other on the protein level in U251 cells which resulted in cell apoptosis inhibition.Mechanism study showed that there was an interaction between TRIM21 and TRIM8 in U251 cells,that interaction furtherly activated ubiquitin-proteasome-dependent degradation pathway through ubiquitination.Conclusion Negatively mutual regulation between TRIM21 and TRIM8 is achieved through ubiquitination modification,following ubiquitin-proteasome-dependent degradation.Our results suggested that it is important to maintain a balance between TRIM21 and TRIM8 on protein level,that is,three might exist an E3 ubiquitin ligase protein homeostasis.The homeostasis disorder will be highly related to the tumorigenesis.

关 键 词：TRIM21 TRIM8 相互调控 泛素化 E3稳态 胶质瘤 

分 类 号：Q51[生物学—生物化学] R753[医药卫生—皮肤病学与性病学]