药学学报, 2019, 54(6): 1026-1035
引用本文:
周丹丹, 余娇娇, 胡卓伟, 花芳. TRIM25增强EGFR稳定性及信号促进肺癌发展[J]. 药学学报, 2019, 54(6): 1026-1035.
ZHOU Dan-dan, YU Jiao-jiao, HU Zhuo-wei, HUA Fang. TRIM25 enhances EGFR stability and signaling activity to promote lung cancer progression[J]. Acta Pharmaceutica Sinica, 2019, 54(6): 1026-1035.

TRIM25增强EGFR稳定性及信号促进肺癌发展
周丹丹, 余娇娇, 胡卓伟, 花芳
中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 新药作用机制研究与药效评价北京市重点实验室(BZ0150), 北京 100050
摘要:
表皮生长因子受体(epidermal growth factor receptor,EGFR)基因是非小细胞肺癌(non-small cell lung cancer,NSCLC)中最常见的驱动基因之一,尤其EGFR突变和扩增都参与了肺癌的恶性进程。本文旨在探究E3泛素连接酶TRIM25(tripartite motif 25)对肺癌发生发展的作用及分子机制。应用CCK-8和Transwell实验评价TRIM25对肺癌细胞增殖和侵袭能力的影响,结果发现敲低TRIM25,显著抑制了细胞的增殖(抑制率为34%)和侵袭(抑制率为42%);采用基因集富集分析、免疫印迹和免疫组化法分析TRIM25对EGFR及其下游信号活性的影响,结果显示,TRIM25不仅上调了EGFR的表达水平,而且促进了EGFR信号活化;利用免疫共沉淀、实时定量PCR以及放线菌酮(cycloheximide,CHX)抑制蛋白质合成实验,初步探究TRIM25上调EGFR信号的分子机制。研究结果表明,TRIM25主要通过促进EGFR第63位赖氨酸位点发生泛素化修饰,进而增加EGFR蛋白稳定性、上调EGFR下游信号活性;而恢复EGFR蛋白表达后可逆转敲低TRIM25对肺癌细胞A549、H1975增殖和侵袭的抑制作用,在A549细胞中,细胞增殖率增加了1.5倍,侵袭率提高了1.6倍;同样,在H1975细胞中,细胞增殖率增加了2倍,侵袭率提高了1.7倍。以上研究结果表明,TRIM25通过维持EGFR信号持续活化,促进肺癌细胞的增殖和侵袭。本研究中所用的人肺癌组织由中国医学科学院肿瘤医院的肺癌患者提供,根据《赫尔辛基宣言》,已取得所有肺癌患者的知情同意。该研究得到了中国医学科学院肿瘤医院伦理委员会的批准。
关键词:    TRIM25      表皮生长因子受体      K63位泛素化      蛋白稳定性     
TRIM25 enhances EGFR stability and signaling activity to promote lung cancer progression
ZHOU Dan-dan, YU Jiao-jiao, HU Zhuo-wei, HUA Fang
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study(BZ0150), Institute of Meteria Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Abstract:
Mutation and amplification of epidermal growth factor receptor (EGFR), one of the most important driver gene, are both reported to participate in the regulation of lung cancer development and progression. Here we investigated the effect and molecular mechanism of tripartite motif 25 (TRIM25) in the regulation of development of lung cancer. CCK-8 and Transwell assays were used to explore the tumor-promoting effect of TRIM25. Results showed that knockdown of TRIM25 significantly inhibited cell proliferation (34% inhibition rate) and invasion (42% inhibition rate). Gene set enrichment analysis (GSEA), Western blot and immunohistochemistry were adopted to detect the effect of TRIM25 on EGFR expression and its downstream signal activity. The results explained that TRIM25 not only up-regulated the expression level of EGFR, but also promoted EGFR signal activation. Co-immunoprecipitation, real-time PCR and cycloheximide (CHX) inhibit protein degradation assays were employed to explore the molecular mechanism of TRIM25 in regulating EGFR stability. Preliminary exploration results indicate that TRIM25 increases the expression level of EGFR and activates its downstream signaling activity through promoting K63-linked ubiquitination of EGFR. Restoration of EGFR expression rescues the phenotype of TRIM25 depletion. In A549 cells, overexpression of EGFR increased cell proliferation rate 1.5-fold and invasion rate 1.6-fold compared with knockdown of TRIM25 cells. Similarly, in H1975 cells, cell proliferation rate was enhanced 2-fold and invasion rate was improved 1.7-fold. These data suggest that TRIM25 promotes lung cancer development via maintaining EGFR stability and continuous EGFR signaling activation. The human lung cancer tissues were obtained from lung cancer patients at Cancer Hospital Chinese Academy of Medical Sciences. Informed consent was obtained from all participants in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of the Cancer Hospital Chinese Academy of Medical Sciences.
Key words:    tripartite motif 25    epidermal growth factor receptor    K63-linked ubiquitination    protein stability   
收稿日期: 2019-04-17
DOI: 10.16438/j.0513-4870.2019-0289
基金项目: 国家自然科学基金面上资助项目(81472717,81673474);北京市自然科学基金面上资助项目(7162133);中国医学科学院医学与健康科技创新工程(2016-I2M-1-007).
通讯作者: 花芳
Email: huafang@imm.ac.cn
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