目的：探究乌头酸酶2（ACO2）发生核转位的条件和作用机制并探讨ACO2核转位对肝癌细胞PLC/PRF/5增殖、迁移和侵袭的影响。方法：借助Human Protein Atlas（HPA）数据库分析ACO2在人肝癌组织中的表达与亚细胞定位；利用NLStradamus网站预测ACO2的潜在核定位信号（NLS），并通过基因截短技术构建NLS缺失的ACO2基因（ACO2 ?NLS）；将野生型（ACO2WT）及突变型（ACO2ΔNLS）质粒转染至人肝癌PLC/PRF/5细胞，分别在常氧及低氧（1% O?）条件下培养；采用免疫荧光染色技术，分析低氧条件影响ACO2入核的机制；通过EdU染色、划痕实验及Transwell实验评估PLC/PRF/5细胞的增殖、迁移与侵袭能力；借助WB检测胞核和胞质中ACO2的表达水平以及迁移、侵袭相关蛋白MMP2与MMP9表达水平的变化。结果：HPA数据库分析显示，ACO2在肝癌细胞的细胞核和细胞质中均呈高表达；免疫荧光染色和WB结果显示，低氧能够以NLS依赖的方式显著诱导ACO2发生核转位（均P < 0.01）；功能学实验显示，低氧诱导的ACO2核转位能进一步显著促进PLC/PRF/5细胞的增殖、迁移（均P < 0.05）和侵袭（均P < 0.05）能力。WB实验表明，ACO2核转位可上调迁移、侵袭相关蛋白MMP2与MMP9的表达水平（均P < 0.05）。结论：低氧能以NLS依赖的方式诱导ACO2发生核转位，从而增强PLC/PRF/5细胞的增殖、迁移和侵袭能力。

Objective: To investigate the conditions and mechanisms by which hypoxia induced the nuclear translocation of aconitase 2 (ACO2) and to explore the effects of ACO2 nuclear translocation on the proliferation, migration, and invasion of hepatocellular carcinoma PLC/PRF/5 cells. Methods: The expression and subcellular localization of ACO2 in human liver cancer tissues were analyzed using the Human Protein Atlas (HPA) database. The potential nuclear localization signal (NLS) of ACO2 was predicted by the NLStradamus website, and an NLS-deficient mutant gene (ACO2ΔNLS) was constructed using genetic engineering techniques. Wild-type (ACO2WT) and mutant (ACO2ΔNLS) plasmids were transfected into human hepatocellular carcinoma PLC/PRF/5 cells, and cells in each transfection group were then cultured under either normoxic or hypoxic (1% O?) conditions. The mechanisms underlying hypoxia-induced nuclear translocation of ACO2 were examined by immunofluorescence staining. The proliferation, migration, and invasion abilities of PLC/PRF/5 cells were assessed using EdU staining, wound healing assay, and Transwell invasion assay. The expression levels of ACO2 in nuclear and cytoplasmic fractions, as well as the levels of migration-and invasion-related proteins MMP2 and MMP9, were detected by WB. Results: HPA database analysis revealed high expression of ACO2 in both the nucleus and cytoplasm of liver cancer cells. Immunofluorescence staining and WB assay results demonstrated that hypoxia significantly induced the nuclear translocation of ACO2 in an NLS-dependent manner (all P < 0.01). Functional experiments showed that hypoxia-induced nuclear translocation of ACO2 further significantly enhanced the proliferation, migration (all P < 0.05), and invasion (all P < 0.05) abilities of PLC/PRF/5 cells. WB analysis indicated that ACO2 nuclear translocation up-regulated the expression levels of migration-and invasion-related proteins MMP2 and MMP9 (all P < 0.05). Conclusion: Hypoxia could induce the nuclear translocation of ACO2 in an NLS-dependent manner, thereby promoting the proliferation, migration, and invasion abilities of PLC/PRF/5 cells.

重庆市教育委员会科学技术研究项目（KJZD-M202202701）；重庆市教委“天然药物抗肿瘤”创新研究（CXQT20030）；三峡库区本土药材筛选与开发（cstc2022ycjh-bgzxm0226）；重庆市科技局项目，三峡库区道地药材开发利用重庆市重点实验室开放课题重点项目（KFKT2022002）