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p LKO. 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。
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.
作者
陶守松
任广明
尹荣华
杨晓明
马文兵
葛志强
TAO Shou-song;REN Guang-ming;YIN Rong-hua;YANG Xiao-ming;MA Wen-bing;GE Zhi-qiang(Department of Pharmaceutical Engineering,Tianjin University,Tianjin 300072,China;Beijing Institute of Lifeomics,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Institute of Health Service and Blood Academy of Military Medical Sciences,Beijing 100850,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2021年第5期1-7,共7页
China Biotechnology
基金
国家自然科学基金青年科学基金(82001666)资助项目。
