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. 2025 Mar 20;45(3):470-478.
doi: 10.12122/j.issn.1673-4254.2025.03.04.

[METTL3-mediated m6A modification promotes FOXO3 expression and anthracycline resistance in acute myeloid leukemia cells through autophagy regulation]

[Article in Chinese]
Xiawei Zhang  1   2 , Jingjing Yang  1   2 , Yanan Wen  1   2 , Qingyang Liu  1   2 , Liping Dou  1 , Chunji Gao  1
Affiliations

[METTL3-mediated m6A modification promotes FOXO3 expression and anthracycline resistance in acute myeloid leukemia cells through autophagy regulation]

[Article in Chinese]
Xiawei Zhang et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objectives: 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 (RNA-seq) 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 m6A-modified mRNA and mRNA stability of FOXO3 was detected analyzed using MeRIP-qPCR 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 m6A 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 m6A-modified FOXO3 expression levels than their sensitive counterparts. Database analysis, Kaplan-Meier analysis and RT-qPCR results suggested that a high FOXO3 expression was associated with a poor prognosis of AML patients. In anthracycline-resistant 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.

Conclusions: METTL3 promotes FOXO3 expression via m6A modification, and FOXO3-driven autophagy contributes to anthracycline resistance in AML cells by enhancing cell proliferation and suppressing cell apoptosis.

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m6A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 m6A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m6A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m6A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。.

Keywords: FOXO3; METTL3; RNA methylation; acute myeloid leukemia; chemotherapy resistance.

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Figures

图1
图1
AML细胞系测序分析 Fig.1 Sequencing analysis of AML cells. A: KEGG enrichment analysis of differential genes in HL60 cells and HL60/ADR cells. B: KEGG enrichment analysis of differential genes in K562/ADR sh-METTL3 cells and K562/ADR sh-con cells. C: MeRIP-seq methylation modification peak analysis in HL60 cells and HL60/ADR cells.
图2
图2
METTL3与FOXO3表达相关性 Fig.2 Correlation of METTL3 with FOXO3 expression. A: Correlation analysis of METTL3 and FOXO3 expression in 163 AML patients from TCGA. B: RT-qPCR analysis of FOXO3 expression in sh-METTL3 cells and sh-con cells. C, D: Western blotting of FOXO3 levels in sh-con cells and sh-METTL3 cells. E: RT-qPCR analysis of FOXO3 expression in OE-METTL3 cells and OE-con cells. F, G: Western blotting of FOXO3 levels in OE-con cells and OE-METTL3. ADR: Adriamycin; sh-con: control shRNA; sh-METTL3: METTL3 shRNA; OE-con: overexpressed control RNA; OE-METTL3: METTL3 overexpression. *P<0.05, **P<0.01, ****P<0.0001.
图3
图3
METTL3通过m6A修饰调控FOXO3 mRNA稳定性 Fig.3 METTL3-mediated m6A modification enhances stability of FOXO3 mRNA. A: MeRIP-qPCR for detecting expression levels of m6A-modified FOXO3 mRNA in anthracycline-resistant cells and anthracycline-sensitive cells. B: MeRIP-qPCR for detecting expression levels of m6A-modified FOXO3 mRNA in OE-con and OE-METTL3 K562/ADR cells. C: RNA stability assay of FOXO3 mRNA in sh-con vs sh-METTL3 groups. *P<0.05, **P<0.01, ***P<0.001.
图4
图4
FOXO3高表达提示患者不良预后 Fig.4 High FOXO3 expression suggests poor prognosis for patients. A: Analysis of FOXO3 expression levels stratified by cytogenetic prognosis in GSE6891 datasets. B: Survival analysis of 160 AML patients in TCGA. C: RT-qPCR analysis of FOXO3 expression in AML patients. D, E: RT-qPCR (D) and Western blotting (E) of FOXO3 expression in anthracycline-sensitive cells and anthracycline-resistant cells. F, G: RT-qPCR (F) and Western blotting (G) of FOXO3 expression in OE-con and OE-FOXO3 K562/ADR cells. H: Cell proliferation and apoptosis analysis of FOXO3-overexpressing cells. *P<0.05, **P<0.01.
图5
图5
FOXO3通过自噬影响AML蒽环类耐药 Fig.5 FOXO3 affects anthracycline resistance of AML cells by regulating autophagy. A: GO enrichment analysis in HL60 and HL60/ADR cells. B: GO enrichment analysis in sh-con cells and sh-METTL3 cells. C: Western blotting of autophagy flow in K562 cells and K562/ADR cells. D: Western blotting of autophagy flow in OE-con cells and OE-FOXO3 cells. E: Proliferation analysis of K562/ADR cells treated with Baf.A1 (100 nmol/L) for 72 h. F: Proliferation analysis of OE-FOXO3 cells treated with ADR and Baf.A1 (10 nmol/L) for 72 h. **P<0.01, ***P<0.001.
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References

    1. Bhansali RS, Pratz KW, Lai C. Recent advances in targeted therapies in acute myeloid leukemia[J]. J Hematol Oncol, 2023, 16(1): 29. - PMC - PubMed
    1. Pollyea DA, Altman JK, Assi R, et al. . Acute myeloid leukemia, version 3.2023, NCCN clinical practice guidelines in oncology[J]. J Natl Compr Canc Netw, 2023, 21(5): 503-13. - PubMed
    1. Thol F, Ganser A. Treatment of relapsed acute myeloid leukemia[J]. Curr Treat Options Oncol, 2020, 21(8): 66. - PMC - PubMed
    1. DiNardo CD, Erba HP, Freeman SD, et al. . Acute myeloid leukaemia[J]. Lancet, 2023, 401(10393): 2073-86. - PubMed
    1. Stelmach P, Trumpp A. Leukemic stem cells and therapy resistance in acute myeloid leukemia[J]. Haematologica, 2023, 108(2): 353-66. - PMC - PubMed

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