华蟾素通过调控MYH9/USP7/c-MYC通路抑制急性髓系白血病细胞免疫逃逸  

Cinobufacini Inhibits Immune Escape of Acute Myeloid Leukemia Cells Through Regulating and Controlling MYH9/USP7/c-MYC Pathway

作　　者：黄蓉 刘凯[1] 郝敬全[1] 王理槐[2] 甘卓 HUANG Rong;LIU Kai;HAo Jing-Quan;WANG Li-Huai;GAN Zhuo(Dept.of Hematology and Oncology,The First Hospital of Hunan University of Chinese Medicine,Changsha 410007 Hunan,China;Dept.of Oncology,The First Hospital of Hunan University of Chinese Medicine,Changsha 410007 Hunan,China;Dept.of Nuclear Medicine,The First Hospital of Hunan University of Chinese Medicine,Changsha 410007 Hunan,China)

机构地区：[1]湖南中医药大学第一附属医院血液肿瘤科,湖南长沙410007 [2]湖南中医药大学第一附属医院肿瘤科,湖南长沙410007 [3]湖南中医药大学第一附属医院核医学科,湖南长沙410007

出　　处：《广州中医药大学学报》2024年第5期1298-1306,共9页Journal of Guangzhou University of Traditional Chinese Medicine

基　　金：湖南省自然科学基金资助项目(编号:2021JJ30525);湖南省临床医疗技术创新引导项目(编号:2022SK51405)。

摘　　要：【目的】探讨华蟾素调控肌球蛋白重链9(MYH9)/泛素特异性蛋白酶7(USP7)/骨髓细胞瘤病毒癌基因(c-MYC)通路对急性髓系白血病(AML)细胞免疫逃逸的影响。【方法】(1)体内实验:建立裸鼠异种移植瘤模型,评估华蟾素对AML细胞在体内生长和免疫逃逸的影响。(2)体外实验:使用不同浓度的华蟾素处理人AML细胞株HL-60,细胞计数试剂盒8(CCK-8)法检测细胞活力,Transwell实验检测细胞侵袭能力。将HL-60细胞与活化的CD8^(+)T细胞共培养,流式细胞术检测CD8^(+)T细胞表面标志物CD25的表达,酶联免疫吸附分析(ELISA)检测共培养上清液中细胞因子[白细胞介素2(IL-2)和干扰素(IFN-γ)]的水平,CytoTox96非放射性细胞毒性分析评估CD8^(+)T细胞对HL-60细胞的毒性。Western Blot法检测MYH9、USP7和c-MYC的蛋白表达,免疫共沉淀(Co-IP)法检测MYH9、USP7和泛素化之间的相互作用。转染MYH9过表质粒,验证华蟾素在AML中的作用机制。【结果】华蟾素抑制裸鼠移植瘤生长,增强CD8^(+)T细胞抗肿瘤的能力。华蟾素浓度依赖性地抑制HL-60细胞活力、侵袭。华蟾素处理后上调CD8^(+)T细胞表面标志物CD25的表达,同时还上调IL-2和IFN-γ水平。华蟾素增强CD8^(+)T细胞对HL-60细胞的毒性。华蟾素抑制HL-60细胞MYH9、USP7和c-MYC的蛋白表达,MYH9通过募集USP7促进c-MYC去泛素化,华蟾素抑制MYH9介导的c-MYC去泛素化。【结论】华蟾素可通过抑制MYH9的表达进而减少去泛素化酶USP7对c-MYC的募集,促进c-MYC泛素化降解,从而抑制AML细胞免疫逃逸。To investigate the effect of cinobufacini on immune escape of acute myeloid leukemia(AML)by regulating myosin heavy chain 9(MYH9)/ubiquitin-specific protease 7(USP7)/cellular-myelocytomatosis viral oncogene(c-MYC)pathway.Methods(1)In vivo experiment:a nude mouse xenograft tumor model was established to evaluate the effect of cinobufotalin on the growth and immune escape of AML cells in vivo.(2)In vitro experiments:human AML cell line HL-60 was treated with different concentrations of cinobufacini,cell viability was detected by cell counting kit 8(CCK-8),and HL-60 cell invasion was detected by Transwell assay.HL-60 cells were co-cultured with activated CD8^(+)T cells,the expression of CD25,the surface marker of CD8^(+)T cells,was detected by flow cytometry,the levels of cytokines[interleukin-2(IL-2)and interferon(IFN-γ)]in the coculture supernatant were detected by enzyme-linked immunosorbent assay(ELISA).CytoTox96 non-radioactive cytotoxicity assay was used to evaluate the cytotoxicity of CD8^(+)T cells to HL-60 cells.The protein expressions of MYH9,USP7 and c-MYC in HL-60 cells were detected by Western Blot.The interaction between MYH9,USP7 and ubiquitination was detected by co-immunoprecipitation(Co-IP)assay.The MYH9 overexpression plasmid was tranfected to verify the mechanism of cinobufacini in AML.Results Cinobufacini treatment inhibited xerograft tumor growth in nude mice and enhanced the anti-tumor ability of CD8^(+)T cells.Cinobufacini treatment inhibited HL-60 cell viability and invasion in a concentration-dependent manner.Cinobufacini treatment up-regulated the expression of CD25,a surface marker of CD8^(+)T cells,and also up-regulated the levels of IL-2 and IFN-γ.Cinobufotalin enhanced the toxicity of CD8^(+)T cells to HL-60 cells.Cinobufacini inhibits the protein expressions of MYH9,USP7 and c-MYC in HL-60 cells.MYH9 promotes c-MYC deubiquitination by recruiting USP7,but cinobufacini inhibits MYH9-mediated c-MYC deubiquitination.Conclusion Cinobufacini can reduce the recruitment of c-MYC by deubiqui

关 键 词：华蟾素 急性髓系白血病 免疫逃逸 MYH9/USP7/c-MYC通路 裸鼠 HL-60细胞 

分 类 号：R285.5[医药卫生—中药学]