JIB-04通过抑制组蛋白赖氨酸去甲基化酶4表达来调控MDM2/P53/SLC7A11/GPX4轴诱导肝癌细胞发生铁死亡  

JIB-04 Regulates the MDM2/P53/SLC7A11/GPX4 Signaling Axis to Induce Ferroptosis in Hepatocellular Carcinoma Cells by Inhibiting KDM4C Expression

作　　者：李健 金俊豪 金艳花 LI Jian;JIN Jun-Hao;JIN Yan-Hua(Department of Cell Biology and Medical Genetics,College of Medicine,Yanbian University,Yanji 133002,Jilin,China)

机构地区：[1]延边大学医学院细胞生物学与医学遗传学教研室,延吉吉林133002

出　　处：《中国生物化学与分子生物学报》2024年第8期1144-1152,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.81660494)资助。

摘　　要：JIB-04(Jumonji histone demethylase inihibitor, JIB-04)是一种泛组蛋白赖氨酸去甲基化酶抑制剂,可抑制多种肿瘤发生发展,但其具体机制仍不清楚。本文以肝癌细胞HepG2和Huh7为研究对象,探讨了JIB-04对肝癌细胞的增殖影响,并揭示其可能的分子机制。CCK-8和EDU检测细胞增殖实验显示,JIB-04明显抑制肝癌细胞HepG2和Huh7活力,且具有浓度依赖性,其半数抑制浓度分别为0.7689μmol/L及0.7392μmol/L。流式细胞术检测细胞内ROS水平变化,结果显示,JIB-04可显著激活细胞内ROS的累积。通过谷胱甘肽(glutathione, GSH)检测试剂盒和脂质过氧化物检测试剂盒分别检测细胞内GSH和脂质过氧化物丙二醛(malondialdehyde, MDA)水平发现,在2μmol/L JIB-04处理下,HepG2和Huh7细胞内GSH分别下降88.4%和80.7%,而脂质过氧化物分别升高4.75倍和9.25倍。通过qRT-PCR和Western印迹分析发现,JIB-04显著降低铁死亡相关因子GPX4和SLC7A11的mRNA水平和蛋白质水平,同时铁死亡相关通路蛋白质MDM2明显下调,P53蛋白水平明显上调。机制研究显示,JIB-04可以使赖氨酸特异性去甲基化酶KDM4C的蛋白质水平下调约58%和51%。通过ChIP检测发现,在JIB-04处理肝癌细胞后MDM2基因启动子区域H3K9me3分别提高了2.19倍和2.14倍,同时qRT-PCR证明,JIB-04处理肝癌细胞后MDM2 mRNA水平显著下调。综上所述,本研究初步揭示了JIB-04可下调特异性去甲基化酶KDM4C的表达,进而影响MDM2基因启动子区域H3K9me3甲基化水平抑制MDM2基因表达,减少MDM2蛋白与P53蛋白结合,P53蛋白水平表达上调,同时,铁死亡相关蛋白质SLC7A11和GPX4的表达下调,导致细胞内GSH耗竭、ROS与脂质过氧化物积累,最终导致细胞发生铁死亡。It has been reported that Jumonji histone demethylase inihibitor(JIB-04)inhibits the occurrence and development of tumors,but the specific mechanism is still unclear.In this paper,hepatocellular carcinoma cells HepG2 and Huh7 were used as the models,and the effect of JIB-04 on the proliferation of hepatocellular carcinoma was explored and its mechanism was explained.CCK-8 assays and EDU staining assays showed that JIB-04 reduced the proliferation ability of HepG2 and Huh7 cells in a concentration-dependent manner,with the half-inhibitory concentrations being 0.7689μmol/L and 0.7392μmol/L,respectively.Flow cytometry analysis showed that JIB-04 could significantly activate the accumulation of ROS in cells.The levels of intracellular GSH and lipid peroxide MDA were detected by glutathione(GSH)detection kit and lipid peroxide malondialdehyde(MDA)detection kit,respectively.It was found that under 2μmol/L JIB-04 treatment,the intracellular GSH of HepG2 and Huh7 cells decreased by 88.4%and 80.7%,respectively.Lipid peroxides increased 4.75-fold and 9.25-fold,respectively.qRT-PCR and Western blotting results showed that JIB-04 could significantly reduce the mRNA levels and protein levels of ferroptosis related factors GPX4 and SLC7A11,while ferroptosis related pathway protein MDM2 was significantly down-regulated and P53 protein was significantly up-regulated.Mechanistic analysis found that JIB-04 reduced histone demethylase KDM4C protein levels by about 58%and 51%.A chromatin immunoprecipitation assay showed that Tri-methylation level of histone H3 at lysine 9 at the promoter region of the MDM 2 gene,was increased by 2.19-fold and 2.14-fold respectively in JIB-04 treated hepatocellular carcinoma cells.Meanwhile,qRT-PCR proved that MDM 2 mRNA level was significantly down-regulated in hepatocellular carcinoma cells after JIB-04 treatment.In summary,this study initially reveals that JIB-04 could downregulate the expression of the specific demethylase KDM4C,and then affect the methylation level of H3K9me3 in the promoter

关 键 词：铁死亡 组蛋白赖氨酸去甲基化酶4 肝细胞癌 Jumonji组蛋白去甲基化酶抑制剂 

分 类 号：Q7[生物学—分子生物学]