RNA m5C甲基转移酶NSUN2调控慢性髓细胞白血病细胞生长和伊马替尼反应性的研究
RNA m5C methyltransferase NSUN2 modulates the growth and imatinib response of chronic myeloid leukemia cells
於志炜 1张秀艳 1赵昀1
作者信息
- 1. 苏州大学唐仲英医学研究院，江苏苏州，215123
- 折叠
摘要
目的：慢性髓细胞白血病（Chronoc Myeloid Leukemia，CML）是一类由BCR-ABL融合基因驱动的髓系增殖性肿瘤。酪氨酸激酶抑制剂（tyrosine kinase inhibitor, TKI）显著改善了患者预后，但耐药问题仍是临床治疗的主要挑战。NSUN2是RNA分子m?C修饰的关键甲基转移酶，与多种肿瘤的发生和进展密切相关。然而，NSUN2在CML中的作用尚不明确。本文旨在探讨NSUN2在CML细胞生长和伊马提尼反应性中的作用。
方法：（1）通过公共数据库分析NSUN2在CML中的表达特征，并采用RT-qPCR检测正常供体及CML患者CD34?细胞中NSUN2的表达水平。（2）利用RNAi介导的基因沉默技术，研究NSUN2对K562细胞m?C修饰丰度、增殖及集落形成能力的影响。（3）在BaF3/BCR-ABL细胞中沉默Nsun2，评估其对m?C修饰水平、增殖及集落形成能力的影响。（4）构建NSUN2野生型（NSUN2-WT）及酶活缺失突变体（NSUN2-MUT），通过回补实验验证NSUN2对K562细胞上述表型的调控是否依赖其甲基转移酶活性。（5）在K562细胞中沉默NSUN2并分别回补NSUN2-WT或NSUN2-MUT，并在BaF3/BCR-ABL细胞中沉默Nsun2，采用CCK-8法检测各组细胞对伊马替尼的敏感性变化。（6）对NSUN2沉默的K562细胞进行转录组测序（RNA-seq），对差异表达基因进行KEGG通路富集分析及GSEA分析，并选取部分差异基因通过RT-qPCR进行表达验证。
结果：（1）NSUN2在CML中显著高表达，CML患者CD34?细胞中NSUN2的mRNA水平显著高于正常供体。（2）沉默NSUN2显著抑制K562细胞的m?C修饰水平、增殖及集落形成能力。（3）沉默Nsun2显著抑制BaF3/BCR-ABL细胞的m?C修饰水平、增殖及集落形成能力。（4）在NSUN2敲低的K562细胞中回补NSUN2-WT可有效恢复m?C修饰水平、增殖及集落形成能力，而回补NSUN2-MUT则无此效应。（5）沉默NSUN2增强K562细胞对伊马替尼的敏感性，回补NSUN2-WT而非NSUN2-MUT可逆转这一效应；沉默Nsun2增强BaF3/BCR-ABL细胞对伊马替尼的敏感性（6）转录组测序（RNA-seq）联合基因集富集分析（GSEA）表明，NSUN2沉默后氨基酸代谢相关通路显著富集；RT-qPCR验证结果与测序数据高度一致，进一步证实了该通路的表达调控特征。
Abstract
Objectives: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm driven by the BCR-ABL fusion oncogene. Although tyrosine kinase inhibitors (TKIs) have significantly improved patient outcomes, drug resistance remains a major clinical challenge. NSUN2 is a key methyltransferase responsible for RNA m?C modification and has been closely implicated in the development and progression of various tumors. However, its role in CML remains unclear. This study aims to investigate the function of NSUN2 in CML cell growth and response to imatinib.
Methods: (1) The expression profile of NSUN2 in CML was analyzed using public databases, and its expression level was validated by RT-qPCR in CD34? cells from normal donors and CML patients. (2) RNA interference (RNAi)-mediated gene silencing was employed to assess the effects of NSUN2 knockdown on m?C modification abundance, proliferation, and colony formation in K562 cells. (3) Nsun2 was silenced in BaF3/BCR-ABL cells to evaluate its impact on m?C modification levels, proliferation, and colony formation. (4) Wild-type NSUN2 (NSUN2-WT) and a methyltransferase-dead mutant (NSUN2-MUT) were constructed, and rescue experiments were performed to determine whether NSUN2 regulates the above phenotypes in a methyltransferase activity-dependent manner. (5) NSUN2-knockdown K562 cells were rescued with NSUN2-WT or NSUN2-MUT, and Nsun2 was silenced in BaF3/BCR-ABL cells; the sensitivity of these cells to imatinib was then assessed by CCK-8 assay. (6) RNA sequencing (RNA-seq) was performed on NSUN2-knockdown K562 cells, followed by KEGG pathway enrichment analysis and GSEA. Selected differentially expressed genes were further validated by RT-qPCR.
Results: (1) NSUN2 was significantly overexpressed in CML, and its mRNA level was markedly higher in CD34? cells from CML patients than in those from normal donors. (2) NSUN2 silencing significantly reduced m?C modification levels, suppressed proliferation, and impaired colony formation in K562 cells. (3) Nsun2 silencing similarly decreased m?C modification levels, proliferation, and colony formation in BaF3/BCR-ABL cells. (4) Rescue of NSUN2-WT, but not NSUN2-MUT, effectively restored m?C modification levels, proliferation, and colony formation in NSUN2-knockdown K562 cells. (5) NSUN2 knockdown enhanced the sensitivity of K562 cells to imatinib, an effect that was reversed by rescue with NSUN2-WT but not NSUN2-MUT; similarly, Nsun2 silencing increased imatinib sensitivity in BaF3/BCR-ABL cells. (6) RNA-seq and GSEA revealed significant enrichment of tumor-related pathways following NSUN2 silencing, with changes in the expression of amino acid metabolism-related genes being highly consistent with the sequencing data.关键词
慢性髓细胞白血病/5-甲基胞嘧啶/NSUN2/伊马替尼Key words
CML/m?C modification/NSUN2/Imatinib引用本文复制引用
於志炜,张秀艳,赵昀.RNA m5C甲基转移酶NSUN2调控慢性髓细胞白血病细胞生长和伊马替尼反应性的研究[EB/OL].(2026-04-29)[2026-04-30].http://www.paper.edu.cn/releasepaper/content/202604-211.学科分类
分子生物学/生物化学/生物科学研究方法、生物科学研究技术
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