WDHD1 Leads to Cisplatin Resistance by Promoting MAPRE2 Ubiquitination in Lung Adenocarcinoma
WDHD1 Leads to Cisplatin Resistance by Promoting MAPRE2 Ubiquitination in Lung Adenocarcinoma
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WDHD1 通过促进肺腺癌中 MAPRE2 泛素化导致顺铂耐药
DOI：
10.3389/fonc.2020.00461
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发表时间：
2020-04-24
影响因子：
4.7
通讯作者：
Cao, Ke
中科院分区：
文献类型：
--
作者：
Gong, Lian;Xiao, Mengqing;Cao, Ke
泛素连接酶已显示调节药物敏感性。本研究旨在探讨泛素连接酶WD重复序列和HMG-box DNA结合蛋白1（WDHD 1）在肺腺癌顺铂敏感性调节中的作用。全局蛋白质组的定量分析确定LUAD A549细胞和顺铂耐药株A549/DDP之间的差异蛋白质表达。公共数据库揭示了泛素连接酶表达与LUAD患者预后的关系。实时定量聚合酶链反应和蛋白质印迹法用于估计WDHD 1表达水平。对公共数据库的分析预测了WDHD 1的底物。Western blotting检测WDHD 1对微管相关蛋白RP/EB家族成员2（MAPRE 2）和DIFFERENTIAL的影响。MAPRE 2的功能分析证实了WDHD 1和MAPRE 2之间的相互作用，以及相互作用的位点，通过甲基噻唑基-四氮唑测定和流式细胞术，免疫沉淀，蛋白质稳定性和免疫荧光。细胞和动物实验证实了WDHD 1和MAPRE 2对LUAD中顺铂敏感性的影响。临床数据评价了WDHD 1表达水平对顺铂敏感性的影响。全局蛋白质组的定量分析显示，A549/DDP细胞中的泛素依赖性蛋白催化剂比A549细胞中的更活跃。WDHD 1在A549/DDP细胞中的表达高于A549细胞，敲除WDHD 1可增加A549/DDP细胞对顺铂的敏感性。WDHD 1过表达与LUAD患者的总生存率呈负相关。我们观察到，当WDHD 1被敲除时，MAPRE 2上调。当A549细胞暴露于顺铂时，A549细胞中的MAPRE 2敲除导致细胞活力增加，同时减少凋亡。发现WDHD 1和MAPRE 2在细胞核中相互作用，并且WDHD 1促进MAPRE 2的泛素化。顺铂暴露后，WDHD 1和MAPRE 2敲除组促进细胞增殖和迁移，抑制A549/DDP细胞的凋亡，减少凋亡，并增加肿瘤大小和生长速度在动物实验。免疫组化显示，Ki 67水平增加，而凋亡指标的水平显着下降，在WDHD 1和MAPRE 2敲除组。临床数据证实，WDHD 1过表达与顺铂敏感性呈负相关。因此，泛素连接酶WDHD 1通过促进MAPRE 2泛素化诱导LUAD中的顺铂抗性。
Ubiquitin ligases have been shown to regulate drug sensitivity. This study aimed to explore the role of the ubiquitin ligase WD repeat and HMG-box DNA binding protein 1 (WDHD1) in regulating cisplatin sensitivity in lung adenocarcinoma (LUAD). A quantitative analysis of the global proteome identified differential protein expression between LUAD A549 cells and the cisplatin-resistant strain A549/DDP. Public databases revealed the relationship between ubiquitin ligase expression and the prognosis of patients with LUAD. Quantitative real-time polymerase chain reaction and Western blotting were used to estimate the WDHD1 expression levels. Analysis of public databases predicted the substrate of WDHD1. Western blotting detected the effect of WDHD1 on microtubule-associated protein RP/EB family member 2 (MAPRE2) and DSTN. Functional analysis of MAPRE2 verified the interaction between WDHD1 and MAPRE2, as well as the interacting sites by methyl-thiazolyl-tetrazolium assay and flow cytometry, immunoprecipitation, protein stability, and immunofluorescence. Cell and animal experiments confirmed the effect of WDHD1 and MAPRE2 on cisplatin sensitivity in LUAD. Clinical data evaluated the impact of WDHD1 expression level on cisplatin sensitivity. Quantitative analysis of the global proteome revealed ubiquitin-dependent protein catabolism to be more active in A549/DDP cells than in A549 cells. WDHD1 expression was higher in A549/DDP cells than in A549 cells, and knocking out WDHD1 increased the sensitivity of A549/DDP cells to cisplatin. WDHD1 overexpression negatively correlated with the overall survival of LUAD patients. We observed that MAPRE2 was upregulated when WDHD1 was knocked out. A MAPRE2 knockout in A549 cells resulted in increased cell viability while decreasing apoptosis when the A549 cells exposed to cisplatin. WDHD1 and MAPRE2 were found to interact in the nucleus, and WDHD1 promoted the ubiquitination of MAPRE2. Following cisplatin exposure, the WDHD1 and MAPRE2 knockout groups facilitated cell proliferation and migration, inhibited apoptosis in A549/DDP cells, decreased apoptosis, and increased tumor size and growth rate in animal experiments. Immunohistochemistry showed that Ki67 levels increased, and levels of apoptotic indicators significantly decreased in the WDHD1 and MAPRE2 knockout groups. Clinical data confirmed that WDHD1 overexpression negatively correlated with cisplatin sensitivity. Thus, the ubiquitin ligase WDHD1 induces cisplatin resistance in LUAD by promoting MAPRE2 ubiquitination.