敲低去泛素化酶USP13抑制K562细胞的增殖<sup>*</sup>

doi:10.13523/j.cb.2101041

中国生物工程杂志

2021, Vol. 41

Issue (5): 1-7 DOI: 10.13523/j.cb.2101041

研究报告

敲低去泛素化酶USP13抑制K562细胞的增殖^*

陶守松¹,任广明²,尹荣华²,杨晓明^1,²,马文兵^3,^**(

),葛志强^1,^**(

)

1 天津大学化工学院制药工程系天津 300072
2 中国人民解放军军事科学院军事医学研究院生命组学研究所北京 100850
3 中国人民解放军军事科学院军事医学研究院卫生勤务与血液研究所北京 100850

Knockdown of Deubiquitinase USP13 Inhibits the Proliferation of K562 Cells

TAO Shou-song¹,REN Guang-ming²,YIN Rong-hua²,YANG Xiao-ming^1,²,MA Wen-bing^3,^**(

),GE Zhi-qiang^1,^**(

)

1 Department of Pharmaceutical Engineering, Tianjin University, Tianjin 300072, China
2 Beijing Institute of Lifeomics, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100850, China
3 Institute of Health Service and Blood Academy of Military Medical Sciences, Beijing 100850, China

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摘要：

目的:研究去泛素化酶USP13对人慢性髓系白血病细胞系K562增殖和凋亡的影响,并进行初步的机制探究。方法:构建pLKO.1-shUSP13-GFP慢病毒干涉载体,慢病毒包装后感染并建立稳定敲低USP13的K562细胞株。免疫印迹检测K562细胞中USP13蛋白的敲低效率。流式细胞术分析敲低USP13对K562细胞增殖和凋亡的影响。免疫共沉淀和蛋白质泛素化实验探究USP13调控K562细胞的分子机制。结果:成功构建pLKO.1-shUSP13-GFP慢病毒干涉载体,同时利用慢病毒体系获得稳定敲低USP13的K562细胞株。流式细胞术结果显示,敲低USP13促进K562细胞凋亡、抑制细胞增殖。分子机制研究发现,敲低USP13通过增强c-Myc泛素化进而导致其蛋白质水平降低。结论:初步揭示了USP13调控K562细胞增殖和凋亡的分子机制,为治疗慢性髓系白血病提供了潜在的靶点。

关键词： 人慢性髓系白血病; K562细胞; USP13; 增殖和凋亡; c-Myc

Abstract:

Objective: Study the effect of deubiquitinating enzyme USP13 on the proliferation and apoptosis of human chronic myeloid leukemia K562 cells, and explore the underlying mechanism. Methods: Construction of the pLKO.1-shUSP13-GFP lentiviral interference vector and establishment of the USP13 knockdown K562 cell line using lentivirus. Western blot detected the USP13 knockdown efficiency in K562 cells. Flow cytometry analyzed the effect of USP13 knockdown on the proliferation and apoptosis of K562 cells. Co-immunoprecipitation and protein ubiquitination experiments explored the regulation mechanism of USP13 on K562 cells. Results: The pLKO.1-shUSP13-GFP vector was successfully constructed, and K562 cell line with stable knockdown of USP13 was obtained using the lentiviral system. Flow cytometry results showed that knocking down USP13 promoted K562 cell apoptosis and inhibited cell proliferation. Molecular mechanism studies found that knockdown of USP13 decreases c-Myc level by enhancing its ubiquitination. Conclusions: Data preliminarily revealed the molecular mechanism of USP13 regulating the proliferation and apoptosis of K562 cells, providing a potential target for the treatment of chronic myeloid leukemia.

Key words: Chronic myelogenous leukemia K562 cells USP13 Proliferation and apoptosis c-Myc

收稿日期: 2021-01-27 出版日期: 2021-06-01

ZTFLH:

Q814

基金资助: * 国家自然科学基金青年科学基金(82001666)

通讯作者: 马文兵,葛志强 E-mail: mawenbing003@163.com;gezhiq@tju.edu.cn

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引用本文:

陶守松,任广明,尹荣华,杨晓明,马文兵,葛志强. 敲低去泛素化酶USP13抑制K562细胞的增殖^*[J]. 中国生物工程杂志, 2021, 41(5): 1-7.

TAO Shou-song,REN Guang-ming,YIN Rong-hua,YANG Xiao-ming,MA Wen-bing,GE Zhi-qiang. Knockdown of Deubiquitinase USP13 Inhibits the Proliferation of K562 Cells. China Biotechnology, 2021, 41(5): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2101041 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I5/1

表1 引物名称和序列

图1 USP13在K562细胞中高水平表达

图2 USP13干涉载体的构建及慢病毒包装

图3 USP13干涉的K562稳定株的构建

图4 敲低USP13促进K562细胞凋亡、抑制细胞增殖

图5 敲低USP13通过增强c-Myc泛素化进而导致其蛋白质水平降低

[1]	Harrington P, Radia D, de Lavallade H. What are the considerations for tyrosine kinase inhibitor discontinuation in chronic-phase chronic myeloid leukemia? Expert Review of Hematology, 2020,13(3):213-222. doi: 10.1080/17474086.2020.1717944 pmid: 31952452
[2]	Breccia M, Efficace F, Colafigli G, et al. Tyrosine kinase inhibitor discontinuation in the management of chronic myeloid leukemia: a critical review of the current practice. Expert Review of Hematology, 2020,13(12):1311-1318. doi: 10.1080/17474086.2021.1852924
[3]	Bower H, Björkholm M, Dickman P W, et al. Life expectancy of patients with chronic myeloid leukemia approaches the life expectancy of the general population. Journal of Clinical Oncology, 2016,34(24):2851-2857. doi: 10.1200/JCO.2015.66.2866
[4]	Cumbo C, Anelli L, Specchia G, et al. Monitoring of minimal residual disease (MRD) in chronic myeloid leukemia: recent advances. Cancer Management and Research, 2020,12:3175-3189. doi: 10.2147/CMAR.S232752
[5]	Eick D. Getting to grips with c-Myc. eLife, 2018,7:32010. DOI: 10.7554/elife.32010.
[6]	Liu N, Li P, Zang S L, et al. Novel agent nitidine chloride induces erythroid differentiation and apoptosis in CML cells through c-Myc-miRNAs axis. PLoS One, 2015,10(2):e0116880. doi: 10.1371/journal.pone.0116880
[7]	Scortegagna M, Subtil T, Qi J F, et al. USP13 enzyme regulates Siah2 ligase stability and activity via noncatalytic ubiquitin-binding domains,. Journal of Biological Chemistry, 2011,286(31):27333-27341. doi: 10.1074/jbc.M111.218214 pmid: 21659512
[8]	Zhang J S, Zhang P J, Wei Y K, et al. Deubiquitylation and stabilization of PTEN by USP13. Nature Cell Biology, 2013,15(12):1486-1494. doi: 10.1038/ncb2874
[9]	Man X J, Piao C Y, Lin X Y, et al. USP13 functions as a tumor suppressor by blocking the NF-kB-mediated PTEN downregulation in human bladder cancer. Journal of Experimental & Clinical Cancer Research, 2019,38(1):259.
[10]	Qu Z, Zhang R, Su M, et al. USP13 serves as a tumor suppressor via the PTEN/AKT pathway in oral squamous cell carcinoma. Cancer Management and Research, 2019,11:9175-9183. doi: 10.2147/CMAR
[11]	Huang J, Gu Z L, Chen W, et al. Knockdown of ubiquitin-specific peptidase 13 inhibits cell growth of hepatocellular carcinoma by reducing c-Myc expression. The Kaohsiung Journal of Medical Sciences, 2020,36(8):615-621. doi: 10.1002/kjm2.v36.8
[12]	Zhang S Z, Zhang M Y, Jing Y, et al. Deubiquitinase USP13 dictates MCL1 stability and sensitivity to BH3 mimetic inhibitors. Nature Communications, 2018,9:215. doi: 10.1038/s41467-017-02693-9
[13]	Fang X G, Zhou W C, Wu Q L, et al. Deubiquitinase USP13 maintains glioblastoma stem cells by antagonizing FBXL14-mediated Myc ubiquitination. The Journal of Experimental Medicine, 2017,214(1):245-267. doi: 10.1084/jem.20151673
[14]	Shao S, Li S, Qin Y W, et al. Spautin-1, a novel autophagy inhibitor, enhances imatinib-induced apoptosis in chronic myeloid leukemia. International Journal of Oncology, 2014,44(5):1661-1668. doi: 10.3892/ijo.2014.2313
[15]	Liao Y N, Liu N N, Xia X H, et al. USP10 modulates the SKP2/Bcr-Abl axis via stabilizing SKP2 in chronic myeloid leukemia. Cell Discovery, 2019,5:24. doi: 10.1038/s41421-019-0092-z
[16]	Zhao X S, Fiske B, Kawakami A, et al. Regulation of MITF stability by the USP13 deubiquitinase. Nature Communications, 2011,2:406-414. doi: 10.1038/ncomms1414
[17]	Guo J, Zhang J L, Liang L, et al. Potent USP10/13 antagonist spautin-1 suppresses melanoma growth via ROS-mediated DNA damage and exhibits synergy with cisplatin. Journal of Cellular and Molecular Medicine, 2020,24(7):4324-4340. doi: 10.1111/jcmm.v24.7

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