N-乙酰转移酶10在尤因肉瘤中的促癌功能及其上下游调控分子机制

doi:10.3969/j.issn.1674-8115.2026.09.001

摘要/Abstract

摘要：

目的·探究N-乙酰转移酶10（N-acetyltransferase 10，NAT10）在尤因肉瘤（Ewing sarcoma，EwS）中的表达、功能及其相关调控机制。方法·整合基因表达综合数据库（Gene Expression Omnibus，GEO）、基因型-组织表达数据库（Genotype-Tissue Expression，GTEx）、基因型和表型数据库（Database of Genotypes and Phenotypes，dbGaP）、癌症细胞系百科全书（Cancer Cell Line Encyclopedia，CCLE），及实验室内EwS肿瘤组织与细胞模型、对照正常肌肉组织与人源间充质干细胞（human mesenchymal stem cells，HMSC）模型的转录组及表观基因组数据，对NAT10等相关基因的转录表达与表观调控机制进行分析。通过反转录定量实时荧光聚合酶链式反应（reverse transcription quantitative real-time PCR，RT-qPCR）、蛋白质印迹法（Western blotting，WB）以及双荧光素酶报告基因实验（dual-luciferase reporter assay，DLR）在细胞模型层面进行验证。利用癌症依赖图谱（Cancer Dependency Map，DepMap）数据库及实验室内开展的成簇规律间隔短回文重复序列及其关联蛋白9（clustered regularly interspaced short palindromic repeats-associated protein 9，CRISPR-Cas9）基因文库筛选数据分析EwS细胞对NAT10的生长依赖性，并通过RNA干扰（RNA interference，RNAi）实验进行验证。整合A673细胞的N⁴-乙酰胞苷测序（ac⁴C sequencing，ac⁴C-seq）与敲低NAT10的RNA测序（RNA sequencing，RNA-seq）数据，筛选受NAT10调控的ac⁴C修饰靶基因，进行基因本体论功能（Gene Ontology，GO）富集分析并针对富集的恶性特征进行功能验证，进一步筛选调控肿瘤生长的关键靶基因。结果·GEO、GTEx、dbGaP、CCLE数据库分析，以及RT-qPCR和WB结果显示，NAT10在EwS肿瘤组织及细胞模型中mRNA和蛋白水平均显著上调。DLR结果显示，EWS RNA结合蛋白1与Friend白血病病毒整合位点1转录因子融合蛋白（EWS RNA-binding protein 1 and Friend leukemia integration site 1 transcription factor fusion protein，EWS-FLI1）通过结合并驱动EwS中特有的NAT10基因增强子促进其转录激活。基因依赖性分析及功能实验结果显示，NAT10在多个EwS细胞模型中呈高度生长依赖性，敲低NAT10显著抑制EwS细胞体外生长与克隆形成。下游促癌分子机制研究表明，EwS细胞模型中NAT10调控的ac⁴C修饰靶基因显著富集于细胞周期、增殖、迁移以及未折叠蛋白反应（unfolded protein response，UPR）等生物学过程，而敲低NAT10引起了细胞周期阻滞、增殖变慢、迁移减弱以及凋亡增强。其中，内着丝粒蛋白（inner centromere protein，INCENP）、驱动蛋白家族成员11（kinesin family member 11，KIF11）、驱动蛋白家族成员14（kinesin family member 14，KIF14）、微小染色体维持蛋白5（minichromosome maintenance complex component 5，MCM5）、非SMC凝缩蛋白Ⅰ复合物G2亚基（non-SMC condensin Ⅰ complex subunit G2，NCAPG2）、DNA聚合酶δ催化亚基1（DNA polymerase δ1，catalytic subunit，POLD1）、DNA拓扑异构酶Ⅱα（DNA topoisomerase Ⅱα，TOP2A）被鉴定为NAT10通过ac⁴C修饰促进其mRNA稳定性，进而调控细胞周期和增殖的关键靶基因。结论·初步揭示“EWS-FLI1—NAT10修饰酶—ac⁴C修饰关键靶基因”调控网络在EwS中的促癌作用与相关机制。

关键词: 尤因肉瘤, EWS RNA结合蛋白1与Friend白血病病毒整合位点1转录因子融合蛋白, N-乙酰转移酶10, mRNA的ac⁴C修饰, 表观遗传失调

Abstract:

Objective ·To investigate the expression, function, and regulatory mechanisms of N-acetyltransferase 10 (NAT10) in Ewing sarcoma (EwS). Methods ·Transcriptomic and epigenomic data from the Gene Expression Omnibus (GEO), the Genotype-Tissue Expression (GTEx), the Database of Genotypes and Phenotypes (dbGaP), and the Cancer Cell Line Encyclopedia (CCLE), as well as in-house EwS tumor tissues and cell models, control normal muscle tissues, and human mesenchymal stem cell (HMSC) models, were integrated to analyze the transcriptional expression and epigenetic regulatory mechanisms of NAT10 and related genes. Validation was performed in cell models using reverse transcription quantitative real-time PCR (RT-qPCR), Western blotting (WB), and dual-luciferase reporter assay (DLR). The dependency of EwS cell growth on NAT10 was analyzed using the Cancer Dependency Map (DepMap) database and in-house clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) library screening data, and further validated by RNA interference (RNAi) experiments. N⁴-acetylcytidine sequencing (ac⁴C-seq) data from A673 cells and RNA sequencing (RNA-seq) data following NAT10 knockdown were integrated to screen for ac⁴C-modified target genes regulated by NAT10. Gene Ontology (GO) enrichment analysis was performed on these targets, followed by functional validation of the enriched malignant features, and subsequently an integrative screening was conducted to identify key target genes involved in regulating tumor growth. Results ·Analyses of the GEO, GTEx, dbGaP, and CCLE database, together with RT-qPCR and WB results, revealed that NAT10 was significantly upregulated at both the mRNA and protein levels in EwS tumor tissues and cell models. DLR results demonstrated that the EWS RNA-binding protein 1 and Friend leukemia integration site 1 transcription factor fusion protein (EWS-FLI1) promotes NAT10 transcriptional activation by binding to and driving the activity of the EwS-specific NAT10 gene enhancer. Gene dependency analysis and functional assays showed that NAT10 exhibited strong growth dependency across multiple EwS cell models, and knockdown of NAT10 significantly inhibited the in vitro growth and colony formation of EwS cells. Mechanistic investigation of downstream pro-tumorigenic pathways revealed that ac⁴C-modified target genes regulated by NAT10 in EwS cell models were significantly enriched in biological processes including the cell cycle, proliferation, migration, and the unfolded protein response (UPR). Knockdown of NAT10 induced cell cycle arrest, reduced proliferation, impaired migration, and enhanced apoptosis. Among these, inner centromere protein (INCENP), kinesin family member 11 (KIF11), kinesin family member 14 (KIF14), minichromosome maintenance complex component 5 (MCM5), non-SMC condensin Ⅰ complex subunit G2 (NCAPG2), DNA polymerase δ1, catalytic subunit (POLD1), and DNA topoisomerase Ⅱα (TOP2A) were identified as key target genes through which NAT10-catalyzed ac⁴C modification promotes mRNA stability, thereby regulating cell cycle and proliferation. Conclusion ·This study preliminarily reveals the pro-tumorigenic role and underlying mechanisms of the “EWS-FLI1-NAT10 modifier enzyme-ac⁴C-modified key target genes” regulatory network in EwS.

Key words: Ewing sarcoma, EWS RNA-binding protein 1 and Friend leukemia integration site 1 transcription factor fusion protein(EWS-FLI1), N-acetyltransferase 10 (NAT10), mRNA ac⁴C modification, epigenetic dysregulation

中图分类号:

R738.1

黄心雨, 莫嘉林, 唐玉杰. N-乙酰转移酶10在尤因肉瘤中的促癌功能及其上下游调控分子机制[J]. 上海交通大学学报（医学版）, 2026, 46(9): 1155-1168.

Huang Xinyu, Mo Jialin, Tang Yujie. Study on the pro-tumorigenic function of N-acetyltransferase 10 in Ewing sarcoma and its upstream and downstream regulatory molecular mechanisms[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026,(9): 1155-1168.

图/表 7

表1 实时荧光定量PCR引物序列（5′→3′）

Tab 1 Primer sequences for real-time quantitative PCR （5′→3′）

Gene	Forward	Reverse
NAT10	GGATTGCCTCAACATCACTCGG	CGTTGGAGGAAAACTTCAGAGGC
EWS-FLI1	GCCAAGCTCCAAGTCAATATAGC	GAGGCCAGAATTCATGTTATTGC
INCENP	AGGCTCCTGAATGTTGAGGTGC	GTGTGCTGTTGGCAATCTCCGT
KIF11	TACAGAAACCACTTAGTAGTGTCC	GAGTTCCTGTGAGAAGCCATCAG
KIF14	GCACTTTCGGAACAAGCAAACCA	ATGTTGCTGGCAGCGGGACTAA
MCM5	GACTTACTCGCCGAGGAGACAT	TGCTGCCTTTCCCAGACGTGTA
NCAPG2	GGAACTGGCATTTGACACGAGC	GCTGCTCTAACAATGGGTGGCT
POLD1	ACTACACGGGAGCCACTGTCAT	GCGTGGTGTAACACAGGTTGTG
TOP2A	GTGGCAAGGATTCTGCTAGTCC	ACCATTCAGGCTCAACACGCTG
GAPDH	TGACTTCAACAGCGACACCCA	CACCCTGTTGCTGTAGCCAAA

表2 shRNA引物序列（5′→3′）

Tab 2 Primer sequences for shRNA （5′→3′）

shRNA ID	Forward	Reverse
shNAT10-1#	CCGGAGGGCCCTCCTTTCCTATAAGCTCGAGCTTATAGGAAAGGAGGGCCCTTTTTTG	AATTCAAAAAAGGGCCCTCCTTTCCTATAAGCTCGAGCTTATAGGAAAGGAGGGCCCT
shNAT10-2#	CCGGCGGCCATCTCTCGCATCTATTCTCGAGAATAGATGCGAGAGATGGCCGTTTTTG	AATTCAAAAACGGCCATCTCTCGCATCTATTCTCGAGAATAGATGCGAGAGATGGCCG

图1 NAT10 在EwS组织及细胞中表达水平的分析与验证Note： A. Multi-cohort analysis from the GEO database. B. Cohort analysis from the GTEx and dbGaP databases. C. Multi-cohort analysis from the GEO database. D. Cohort analysis from GSE184225 and the CCLE databases. E. RNA-seq analysis of NAT10 expression in HMSC and EwS cell lines. F. RT-qPCR validation of NAT10 mRNA expression levels in HMSC and EwS cells (A673 and SKNMC). G. WB validation of NAT10 expression and EWS-FLI1 protein levels in HMSC and EwS cells (A673 and SKNMC). The numbers below the blots indicate the fold changes relative to the A673 group, which were normalized to β-tubulin. FPKM—fragments per kilobase of transcript per million mapped reads.

Fig 1 Analysis and validation of NAT10 expression levels in EwS tissues and cells

图2 基于ChIP-seq、RNA-seq及双荧光素酶报告实验解析EWS-FLI1对 NAT10 的转录调控作用Note： A. RNA-seq analysis of NAT10 expression in A673 and SKNMC cells following EWS-FLI1 knockdown (relative to the shScr group, P<0.001). B. ChIP-seq analysis of EWS-FLI1 binding and H3K27ac enrichment at the NAT10 enhancer (containing 12×GGAA repeats); enhancer-promoter interaction was revealed by H3K27ac HiChIP (GSE133227). Data from EwS cell lines and 3 patient tumor tissues were included. C. RNA-seq analysis of NAT10 expression in HMSCs with ectopic EWS-FLI1 overexpression (EWS-FLI1_OE) or wild-type FLI1 overexpression (FLI1_OE); NAT10 was found to be significantly upregulated in the EWS-FLI1_OE group (P=0.007). D. ChIP-seq analysis of EWS-FLI1 binding and H3K27ac enrichment at the NAT10 enhancer in HMSCs with EWS-FLI1 or wild-type FLI1 overexpression; de novo enhancer signals were confirmed to be induced only by EWS-FLI1. E. A schematic of the dual-luciferase reporter assay: NAT10/CCND1-enhancer fragments were cloned into the pGL3 vector (with miniP), and the resulting constructs were co-transfected with the EWS-FLI1 expression plasmid. F. Dual-luciferase reporter assay showed significant transactivation of the NAT10 enhancer by EWS-FLI1 in 293T cells (P<0.001); the CCND1-enhancer was used as the positive control, and the miniP vector as the negative control. G. RT-qPCR analysis of NAT10 expression in A673 and SKNMC cells following EWS-FLI1 knockdown (relative to the shScr control group); NAT10 expression was significantly downregulated (P<0.001).

Fig 2 Transcriptional regulation of NAT10 by EWS-FLI1 analyzed via ChIP-seq, RNA-seq, and dual-luciferase reporter assays

图3 基于DepMap数据库、CRISPR-Cas9筛选及体外功能实验解析 NAT10 在EwS中的肿瘤依赖性Note： A. The dependency of NAT10 across EwS cell lines harboring distinct fusion types. B. CRISPR-Cas9 library screening in A673 cells was performed, and NAT10 was essential for in vitro proliferation (ranked 115th overall). C. Depletion efficiency of five independent NAT10-targeting sgRNAs in CRISPR-Cas9 library screening. D/E. RT-qPCR validation of NAT10 mRNA knockdown in A673 (D) and SKNMC (E) cells, compared with the shScr group. F/G. WB analysis of NAT10 protein expression in A673 (F) and SKNMC (G) cells after knockdown; the numbers below the blots indicate the fold changes normalized to β-tubulin. H/I. Assessment of cell viability in A673 (H) and SKNMC (I) cells after NAT10 knockdown, compared with the shScr group. J/K. Colony formation assay in A673 (J) and SKNMC (K) cells after NAT10 knockdown, compared with the shScr group (P<0.001).

Fig 3 Evaluation of the dependency of EwS on NAT10via the DepMap database, CRISPR-Cas9 screening, and in vitro functional assays

图4 NAT10 调控的ac4C修饰靶基因鉴定及其功能富集分析Note： A. Volcano plot of differentially expressed genes in A673 cells after NAT10 knockdown. B. Distribution of ac4C modification sites across mRNA regions in A673 cells. C. Venn diagram of NAT10-targeted ac4C-modified genes identified by integrating RNA-seq and ac4C-seq data. D. GO functional enrichment analysis of 318 NAT10-targeted ac4C-modified genes. E‒H. Functional assays in A673 cells after NAT10 knockdown: cell cycle (E), proliferation assessed by EdU (F), apoptosis assessed by Annexin Ⅴ/PI staining (G), and migration assessed by wound healing assay (H), compared with the shScr group.

Fig 4 Identification and functional analysis of NAT10-regulated ac4C -modified genes

图5 鉴定 NAT10 调控的ac4C修饰关键靶基因及其mRNA稳定性验证Note： A. Venn diagram identifying seven tumor-promoting genes. B/C. RNA-seq analysis of seven candidate genes in EwS cells (B) and tissues (C). D. Dependency analysis of seven candidate genes in EwS cell lines. E. Distribution of ac4C modification sites on the mRNAs of the seven candidate genes. F. RNA-seq analysis of the expression of seven candidate genes after NAT10 knockdown in A673 cells, compared with the shScr group. G. RT-qPCR validation of the expression of seven candidate genes after NAT10 knockdown in A673 cells, compared with the shScr group. H. mRNA stability analysis of the seven candidate genes in NAT10-knockdown A673 cells.

Fig 5 Identification of key NAT10-regulated ac4C-modified target genes and validation of their mRNA stability

参考文献 39

[1]	Grünewald T G P, Cidre-Aranaz F, Surdez D, et al. Ewing sarcoma[J]. Nat Rev Dis Primers, 2018, 4: 5.
[2]	Riggi N, Suva M L, Stamenkovic I. Ewing′s sarcoma[J]. N Engl J Med, 2021, 384(2): 154-164.
[3]	Monument M J, Johnson K M, Grossmann A H, et al. Microsatellites with macro-influence in ewing sarcoma[J]. Genes, 2012, 3(3): 444-460.
[4]	Gangwal K, Sankar S, Hollenhorst P C, et al. Microsatellites as EWS/FLI response elements in Ewing′s sarcoma[J]. Proc Natl Acad Sci USA, 2008, 105(29): 10149-10154.
[5]	Tomazou E M, Sheffield N C, Schmidl C, et al. Epigenome mapping reveals distinct modes of gene regulation and widespread enhancer reprogramming by the oncogenic fusion protein EWS-FLI1[J]. Cell Rep, 2015, 10(7): 1082-1095.
[6]	Palombo R, Frisone P, Fidaleo M, et al. The promoter-associated noncoding RNA pncCCND1_B assembles a protein-RNA complex to regulate cyclin D1 transcription in ewing sarcoma[J]. Cancer Res, 2019, 79(14): 3570-3582.
[7]	Dauphinot L, De Oliveira C, Melot T, et al. Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57KIP2 and c-Myc expression[J]. Oncogene, 2001, 20(25): 3258-3265.
[8]	Shi X P, Zheng Y Y, Jiang L L, et al. EWS-FLI1 regulates and cooperates with core regulatory circuitry in Ewing sarcoma[J]. Nucleic Acids Res, 2020, 48(20): 11434-11451.
[9]	Uren A, Tcherkasskaya O, Toretsky J A. Recombinant EWS-FLI1 oncoprotein activates transcription[J]. Biochemistry, 2004, 43(42): 13579-13589.
[10]	Erkizan H V, Uversky V N, Toretsky J A. Oncogenic partnerships: EWS-FLI1 protein interactions initiate key pathways of Ewing′s sarcoma[J]. Clin Cancer Res, 2010, 16(16): 4077-4083.
[11]	Conn E, Hour S, Allega Koen D, et al. Development of an Ewing sarcoma cell line with resistance to EWS-FLI1 inhibitor YK-4-279[J]. Mol Med Rep, 2020, 21(3): 1667-1675.
[12]	Meyers P A, Federman N, Daw N, et al. Open-label, multicenter, phase Ⅰ/Ⅱ, first-in-human trial of TK216: a first-generation EWS:FLI1 fusion protein antagonist in ewing sarcoma[J]. J Clin Oncol, 2024, 42(31): 3725-3734.
[13]	Mohibi S, Chen X B, Zhang J. Cancer the'RBP'eutics-RNA-binding proteins as therapeutic targets for cancer[J]. Pharmacol Ther, 2019, 203: 107390.
[14]	An Y Y, Duan H. The role of m6A RNA methylation in cancer metabolism[J]. Mol Cancer, 2022, 21(1): 14.
[15]	Yu L, Xu H E, Xiong H C, et al. The role of m5C RNA modification in cancer development and therapy[J]. Heliyon, 2024, 10(19): e38660.
[16]	Luo Y J, Yao Y X, Wu P, et al. The potential role of N⁷-methylguanosine (m7G) in cancer[J]. J Hematol Oncol, 2022, 15(1): 63.
[17]	Barbieri I, Kouzarides T. Role of RNA modifications in cancer[J]. Nat Rev Cancer, 2020, 20(6): 303-322.
[18]	Jiang J, Fan Q, Qu H S, et al. Novel prognostic biomarkers, METTL14 and YTHDF2, associated with RNA methylation in Ewing′s sarcoma[J]. Sci Rep, 2022, 12: 7041.
[19]	He F Z, Ren T T, Tang X D. METTL3-modified lncRNA-MALAT1 regulates the molecular axis of miR-124-3p/CDK4 involved in Ewing′s sarcoma[J]. Cell Mol Biol, 2023, 69(6): 193-197.
[20]	Chen Y Q, Jiao X J, Xu B T, et al. Gene signatures of m5C regulators may predict prognosis and immunity status of patients with Ewing′s sarcoma[J]. Int Immunopharmacol, 2025, 159: 114849.
[21]	Gu Z R, Zou L B, Pan X J, et al. The role and mechanism of NAT10-mediated ac⁴C modification in tumor development and progression[J]. MedComm, 2024, 5(12): e70026.
[22]	Arango D, Sturgill D, Alhussaini N, et al. Acetylation of cytidine in mRNA promotes translation efficiency[J]. Cell, 2018, 175(7): 1872-1886.e24.
[23]	Rodrigues P, Bangali H, Ali E, et al. The mechanistic role of NAT10 in cancer: unraveling the enigmatic web of oncogenic signaling[J]. Pathol Res Pract, 2024, 253: 154990.
[24]	Jin C, Wang T, Zhang D S, et al. Acetyltransferase NAT10 regulates the Wnt/β-catenin signaling pathway to promote colorectal cancer progression via ac⁴C acetylation of KIF23 mRNA[J]. J Exp Clin Cancer Res, 2022, 41(1): 345.
[25]	Pan Z P, Bao Y W, Hu M Y, et al. Role of NAT10-mediated ac⁴C-modified HSP90AA1 RNA acetylation in ER stress-mediated metastasis and lenvatinib resistance in hepatocellular carcinoma[J]. Cell Death Discov, 2023, 9(1): 56.
[26]	Zou Y T, Guo S Y, Wen L L, et al. Targeting NAT10 inhibits osteosarcoma progression via ATF4/ASNS-mediated asparagine biosynthesis[J]. Cell Rep Med, 2024, 5(9): 101728.
[27]	Mo J, Tan K, Dong Y, et al. Therapeutic targeting the oncogenic driver EWSR1::FLI1 in Ewing sarcoma through inhibition of the FACT complex[J]. Oncogene, 2023, 42(1): 11-25.
[28]	Galvan B, Ongena L, Bruyr J, et al. Subversion of mRNA degradation pathways by EWSR1: FLI1 represents a therapeutic vulnerability in Ewing sarcoma[J]. Nat Commun, 2025, 16: 6537.
[29]	Liu Y Y, Wang X, Liu Y Y, et al. N⁴-acetylcytidine-dependent GLMP mRNA stabilization by NAT10 promotes head and neck squamous cell carcinoma metastasis and remodels tumor microenvironment through MAPK/ERK signaling pathway[J]. Cell Death Dis, 2023, 14(11): 712.
[30]	Chen X N, Hao Y, Liu Y, et al. NAT10/ac⁴C/FOXP1 promotes malignant progression and facilitates immunosuppression by reprogramming glycolytic metabolism in cervical cancer[J]. Adv Sci, 2023, 10(32): 2302705.
[31]	Matsuhashi A, Ohno T, Kimura M, et al. Growth suppression and mitotic defect induced by JNJ-7706621, an inhibitor of cyclin-dependent kinases and aurora kinases[J]. Curr Cancer Drug Targets, 2012, 12(6): 625-639.
[32]	Turaga S M, Vishwakarma V, Hembluff S L, et al. Inducing mitotic catastrophe as a therapeutic approach to improve outcomes in ewing sarcoma[J]. Cancers, 2023, 15(20): 4911.
[33]	Ayala F R R, Martin J W, Bertuzzzo C S. Replication timing aberration of KIF14 and MDM4/PI3KC 2β alleles and aneuploidy as markers of chromosomal instability and poor treatment response in ewing family tumor patients[J]. Glob Med Genet, 2023, 10(2): 54-62.
[34]	Mao J Y, Shen J, Lu X M, et al. MCM5 is an oncogene of colon adenocarcinoma and promotes progression through cell cycle control[J]. Acta Histochem, 2023, 125(6): 152072.
[35]	Zhan P, Xi G M, Zhang B, et al. NCAPG2 promotes tumour proliferation by regulating G2/M phase and associates with poor prognosis in lung adenocarcinoma[J]. J Cell Mol Med, 2017, 21(4): 665-676.
[36]	Nicolas E, Golemis E A, Arora S. POLD1: central mediator of DNA replication and repair, and implication in cancer and other pathologies[J]. Gene, 2016, 590(1): 128-141.
[37]	Zhang K W, Zheng X Y, Sun Y Q, et al. TOP2A modulates signaling via the AKT/mTOR pathway to promote ovarian cancer cell proliferation[J]. Cancer Biol Ther, 2024, 25(1): 2325126.
[38]	Shi L, Zhang M W, Yang H, et al. NAT10 regulates heart development and function by maintaining the expression of genes related to fatty acid β-oxidation and heart contraction[J]. Cell Death Differ, 2026, 33(2): 358-373.
[39]	Larrieu D, Britton S, Demir M, et al. Chemical inhibition of NAT10 corrects defects of laminopathic cells[J]. Science, 2014, 344(6183): 527-532.