METTL3介导的m^(6)A修饰通过调节自噬促进急性髓性白血病细胞中FOXO3表达及蒽环类药物耐药性  

METTL3-mediated m^(6)A modification promotes FOXO3 expression and anthracycline resistance in acute myeloid leukemia cells through autophagy regulation

作　　者：张夏玮 杨晶晶 温亚男 刘青阳 窦立萍 高春记 ZHANG Xiawei;YANG Jingjing;WEN Yanan;LIU Qingyang;DOU Liping;GAO Chunji(Medical School of Chinese PLA,Beijing 100853,China;Department of Hematology,Fifth Medical Center of Chinese PLA General Hospital,Beijing 100853,China)

机构地区：[1]解放军医学院,北京100853 [2]中国人民解放军总医院第五医学中心血液病医学部,北京100853

出　　处：《南方医科大学学报》2025年第3期470-478,共9页Journal of Southern Medical University

基　　金：国家自然科学基金(82270162,82270224,82170207);北京市自然科学基金(7222175);国家重点研发计划(2023YFC2507800);北京市科技新星交叉合作项目(20230484407);后勤科研自主项目(2023hqzz09);首都卫生发展科研专项(首发2024-2-5063)

摘　　要：目的探讨急性髓系白血病(AML)细胞中叉头盒蛋白3(FOXO3)与甲基转移酶样蛋白3(METTL3)表达关系及FOXO3参与AML化疗耐药的机制。方法采用敲低或过表达METTL3和FOXO3及其对照慢病毒载体,转染AML蒽环类耐药细胞系,获取对照组、敲低组、过表达组细胞。对AML细胞采用甲基化RNA共沉淀和高通量测序技术(MeRIP-seq)和转录组测序(RNA-seq)。TCGA和GSE6891数据库对AML患者临床信息及基因表达数据进行统计分析。RT-qPCR及Western blotting检测FOXO3 mRNA及蛋白的表达水平和FOXO3 mRNA稳定性。流式细胞术和CCK-8分别检测细胞凋亡和增殖能力的变化。MeRIP-qPCR检测m^(6)A修饰FOXO3 mRNA的表达情况。结果蒽环类敏感及耐药AML细胞系和敲低METTL3前后细胞系差异基因均富集在FoxOs通路中,且耐药细胞中FOXO3 m^(6)A修饰明显增加。公共数据库相关性分析显示FOXO3与METTL3表达呈正相关(P<0.01)。Western blotting和RT-qPCR结果提示敲低METTL3后FOXO3表达下降(P<0.05)。MeRIP-qPCR结果提示蒽环类耐药AML细胞中m^(6)A修饰的FOXO3 mRNA表达高于蒽环类敏感AML细胞(P<0.05)。稳定性试验结果提示敲低METTL3后FOXO3 mRNA稳定性降低。公共数据库分析、Kaplan-Meier分析和RT-qPCR结果提示FOXO3与AML患者预后不良相关(P<0.05)。Western blotting和RT-qPCR结果显示,蒽环类耐药细胞中FOXO3的表达明显高于敏感细胞(P<0.05)。体外实验显示过表达FOXO3的AML细胞后细胞增殖更快,凋亡减少(P<0.05)。蒽环类敏感及耐药AML细胞系和敲低METTL3前后细胞系差异基因均富集在自噬相关通路中,抑制自噬增强阿霉素对AML细胞和过表达FOXO3细胞的抗肿瘤作用。结论METTL3可能通过介导的m^(6)A修饰促进FOXO3的表达,进而调节自噬促进蒽环类药物耐药细胞的增殖和抑制凋亡。Objective To investigate the role of METTL3 and FOXO3 in anthracycline resistance in acute myeloid leukemia(AML)cells.Methods Methylated RNA immunoprecipitation sequencing(MeRIP-seq)and transcriptome sequencing(RNAseq)were performed in anthracycline-resistant and sensitive HL60 and K562 cells with lentivirus-mediated knockdown or overexpression of METTL3 and FOXO3.TCGA and GSE6891 datasets were used for analysis of the clinical and gene expression data of AMI patients.FOXO3 expressions at the mRNA and protein levels in the transfected cells were detected with RT-qPCR and Western blotting,and the changes in cell proliferation and apoptosis were evaluated using CCK8 assay and flow cytometry;the expression of m^(6)A-modified mRNA and mRNA stability of FOXO3 was detected analyzed using MeRIPqPCR and RT-qPCR.Functional enrichment analysis of the differential genes in the transfected cells was performed.Results Differential gene analysis in anthracycline-resistant versus sensitive AML cells and in cells with METTL3 knockdown revealed the enrichment in FoxO and autophagy pathways(P<0.05),and the anthracycline-resistant cells showed significantly increased m^(6)A modification of FOXO3.FOXO3 expression was positively correlated with METTL3 expression.METTL3 knockdown significantly reduced FOXO3 mRNA stability and its protein levels in anthracycline-resistant AML cells,which exhibited higher m^(6)A-modified FOXO3 expression levels than their sensitive counterparts.Database analysis,Kaplan-Meier analysis and RTqPCR results suggested that a high FOXO3 expression was associated with a poor prognosis of AML patients.In anthracyclineresistant AML cells expressing higher FOXO3 levels than the sensitive cells,lentivirus-mediated overexpression of FOXO3 significantly enhanced cell proliferation and suppressed cell apoptosis.Inhibiting autophagy using an autophagy inhibitor(Baf.A1)obviously enhanced the inhibitory effect of adriamycin on resistant AMI cells and cells overexpressing FOXO3.Conclusion METTL3 promotes FOXO3 expression

关 键 词：急性髓系白血病 化疗耐药 FOXO3 METTL3 RNA甲基化 

分 类 号：R73[医药卫生—肿瘤]