PSME2和STAT3双重抑制通过调控SLC7A11/GPX4轴诱导食管鳞癌细胞铁死亡

doi:10.12122/j.issn.1673-4254.2026.09.21

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (9): 2230-2241.doi: 10.12122/j.issn.1673-4254.2026.09.21

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PSME2和STAT3双重抑制通过调控SLC7A11/GPX4轴诱导食管鳞癌细胞铁死亡

祁春晖(), 张鹤骞, 吴晓爽, 康永安, 杨静怡, 赵迪, 高社干, 齐义军()

河南科技大学临床医学院//河南科技大学第一附属医院，肿瘤医院//省部共建食管癌防治国家重点实验室//河南省微生态与食管癌防治重点实验室//河南省肿瘤表观遗传实验室，河南洛阳 471003

收稿日期:2026-02-18 出版日期:2026-09-20 发布日期:2026-09-30
通讯作者: 齐义军 E-mail:qch1664037558@163.com;qiqiyijun@163.com
作者简介:祁春晖，在读硕士研究生，E-mail:qch1664037558@163.com
基金资助:
国家自然科学基金(81872037);河南省国际科技合作重点项目(261111520900)

Dual inhibition of PSME2 and STAT3 induces ferroptosis in esophageal squamous cell carcinoma cells by regulating the SLC7A11/GPX4 axis

Chunhui QI(), Heqian ZHANG, Xiaoshuang WU, Yongan KANG, Jingyi YANG, Di ZHAO, Shegan GAO, Yijun QI()

State Key Laboratory of Esophageal Cancer Prevention &Treatment, Henan Key Laboratory of Microbiome and Esophageal Cancer Prevention and Treatment, Henan Provincial Laboratory of Cancer Epigenetics, Cancer Hospital, First Affiliated Hospital, College of Clinical Medicine of Henan University of Science and Technology, Luoyang 471003, China

Received:2026-02-18 Online:2026-09-20 Published:2026-09-30
Contact: Yijun QI E-mail:qch1664037558@163.com;qiqiyijun@163.com
Supported by:
National Natural Science Foundation of China(81872037)

摘要/Abstract

摘要：

目的探究蛋白酶体激活物复合物亚基2（PSME2）沉默联合STAT3抑制剂WP1066诱导食管鳞癌（ESCC）细胞死亡的形式及其分子机制。方法在ESCC细胞（KYSE30， NE6-T）中沉默PSME2并联合WP1066处理。应用不同细胞死亡抑制剂（Ferrostatin-1，Z-VAD-FMK，Necrosulfonamide等）进行表型挽救实验，以鉴别细胞死亡类型；Western blotting检测铁死亡（SLC7A11和GPX4）、凋亡（Cleaved Caspase-3和PARP）、焦亡（GSDME）、坏死性凋亡（RIP）等蛋白质分子表达变化；DC-FHDA和C11-BODIPY荧光探针分别检测细胞内活性氧（ROS）水平和脂质过氧化程度；透射电镜（TEM）观察线粒体超微结构变化。mRNA转录组分析PR171或siPSME2联合WP1066处理的各组细胞间差异表达基因；参考Hallmark及KEGG基因集应用GSEA进行生物学功能富集分析。结果 PSME2和WP1066双重抑制能够诱导ESCC细胞死亡，该致死效应能被铁死亡抑制剂Ferrostatin-1逆转，凋亡（Z-VAD-FMK）次之，但不能被焦亡抑制剂2-Bromohexadecanoic acid和坏死性凋亡抑制剂Necrosulfonamide阻断（P<0.0001）。siPSME2与WP1066联合处理组ESCC细胞显著下调SLC7A11和GPX4蛋白质分子的表达，同时细胞内ROS水平升高和脂质过氧化物积累（P<0.001）。TEM显示双重抑制组ESCC细胞线粒体皱缩、嵴减少、膜密度增高等改变。mRNA转录组分析结果显示，PR171和WP1066联合处理组与siPSME2和WP1066联合处理组具有45个共同差异表达基因集，这些基因集的生物学功能富集于应激反应、氧化还原失衡、蛋白翻译/核糖体功能、代谢、细胞周期等多个特征学通路。结论 PSME2和STAT3双重抑制通过调控SLC7A11/GPX4抗氧化轴，促进脂质过氧化积累诱导ESCC细胞铁死亡。

关键词: 蛋白酶体激活物复合物亚基2, STAT3, 食管鳞癌, 铁死亡, SLC7A11, GPX4

Abstract:

Objective To investigate the form of cell death induced by PSME2 silencing combined with the STAT3 inhibitor WP1066 in esophageal squamous cell carcinoma (ESCC) cells and explore the underlying mechanism. Methods ESCC cell lines KYSE30 and NE6-T with PSME2 silencing were treated with WP1066. Phenotypic rescue experiments were performed to determine the predominant form of cell death using different cell death-related inhibitors. Western blotting was used to detect the expressions of the protein markers of ferroptosis, apoptosis, pyroptosis and necroptosis. Intracellular reactive oxygen species (ROS) levels and lipid peroxidation were assessed using DCFH-DA and C11-BODIPY fluorescent probes, respectively. Mitochondrial ultrastructural changes were observed by transmission electron microscopy (TEM). mRNA transcriptomic analysis was performed to identify differentially expressed genes in cells treated with PR171 or siPSME2 combinedwith WP1066. Gene set enrichment analysis to analyze the enriched biological functions. Results Dual inhibition of PSME2 and STAT3 induced obvious ESCC cell death. This lethal effect was markedly reversed by the ferroptosis inhibitor ferrostatin-1 and attenuated by the apoptosis inhibitor Z-VAD-FMK, whereas 2-bromohexadecanoic acid or necrosulfonamide did not show significant inhibitory effects. The combined treatment with siPSME2 and WP1066 significantly downregulated SLC7A11 and GPX4 protein expressions and increased intracellular ROS levels and lipid peroxide accumulation in ESCC cells. The ESCC cells with dual inhibition showed obvious mitochondrial shrinkage, reduced cristae, and increased membrane density. mRNA transcriptomic analysis identified 45 overlapping differentially expressed genes between the PR171 plus WP1066 group and the siPSME2 plus WP1066 group, enriched in pathways related to stress response, redox imbalance, protein translation/ribosomal function, metabolism, and cell cycle regulation. Conclusion Dual inhibition of PSME2 and STAT3 promotes lipid peroxide accumulation and induces ferroptosis in ESCC cells possibly by regulating the SLC7A11/GPX4 antioxidant axis, indicating a therapeutic strategy targeting both PSME2 and STAT3 for ESCC.

Key words: proteasome activator complex subunit 2, STAT3, esophageal squamous cell carcinoma, ferroptosis, SLC7A11, GPX4

祁春晖, 张鹤骞, 吴晓爽, 康永安, 杨静怡, 赵迪, 高社干, 齐义军. PSME2和STAT3双重抑制通过调控SLC7A11/GPX4轴诱导食管鳞癌细胞铁死亡[J]. 南方医科大学学报, 2026, 46(9): 2230-2241.

Chunhui QI, Heqian ZHANG, Xiaoshuang WU, Yongan KANG, Jingyi YANG, Di ZHAO, Shegan GAO, Yijun QI. Dual inhibition of PSME2 and STAT3 induces ferroptosis in esophageal squamous cell carcinoma cells by regulating the SLC7A11/GPX4 axis[J]. Journal of Southern Medical University, 2026, 46(9): 2230-2241.

图/表 5

图1 铁死亡抑制剂明显逆转了PSME2和STAT3双重抑制诱导的ESCC细胞死亡

Fig.1 Ferroptosis inhibition markedly reverses cell death induced by dual inhibition of PSME2 and STAT3 in esophageal squamous cell carcinoma (ESCC) cells. A, B: Released LDH activity (mU/mL) in ESCC cells after different treatments (A: KYSE30; B: NE6-T). C: Immunofluorescence staining of Calcein-AM/PI and quantitative assessment across different groups (green indicates live cells, and red indicates dead cells; scale bar=275 μm). D, E: Quantitative analyses of the percentage of cell death (%) in ESCC cells after different treatments (D: KYSE30; E: NE6-T). Data are presented as Mean±SD (n=3). **P<0.01,***P<0.001, ****P<0.0001 vs siPSME2+WP1066.

图2 PSME2和STAT3双重抑制下调铁死亡关键靶点分子GPX4和SLC7A11表达

Fig.2 Dual inhibition of PSME2 and STAT3 induces downregulation of ferroptosis-related molecules GPX4 and SLC7A11. A, B: Western blotting of the protein levels of PSME2, PARP, cleaved PARP, caspase-3 and cleaved caspase-3 in KYSE30 and NE6-T cells. C, D: Results of Western blotting of GSDME and RIP in KYSE30 and NE6-T cells. E, F: Results of Western blotting of GPX4 and SLC7A11 in KYSE30 and NE6-T cells. G: GPX4 and SLC7A11 mRNA expressions in KYSE30 cells. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs siControl.

图3 PSME2和STAT3双重抑制诱导ESCC细胞脂质过氧化、Fe²+积累、ROS积累及线粒体超微结构改变

Fig.3 Dual inhibition of PSME2 and STAT3 induces lipid peroxidation, Fe²⁺ accumulation, ROS accumulation, and mitochondrial ultrastructural changes in ESCC cells. A, B: Lipid peroxidation levels measured by C11-BODIPY fluorescence staining in ESCC cells after different treatments (green for oxidation state, and red for reduction state; A: KYSE30; B: NE6-T; Scale bars=10 μm ). C, D: MDA levels (umol/ug) in ESCC cells after indicated treatments (C: KYSE30; D: NE6-T). E: Transmission electron microscopy of ESCC cells after different treatments (Scale bar=5 μm ). F, G: Intracellular ROS detected using DCFH-DA staining in KYSE30 and NE6-T cells (Scale bar=125 μm). H, I: Intracellular Fe²⁺ levels detected using FerroOrange staining in KYSE30 and NE6-T cells (Scale bar=125 μm). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs siControl.

图4 PSME2和STAT3双重抑制降低ESCC细胞糖酵解水平和线粒体有氧呼吸

Fig.4 Dual inhibition of PSME2 and STAT3 reduces glycolytic activity and mitochondrial respiration in ESCC cells. A: ECAR curves showing glycolysis rates of indicated cells after different treatments. B: OCR curves reflecting mitochondrial respiration capacity. C, D: Quantification of basal respiration, ATP production, proton leak, spare respiration capacity, non-glycolytic acidification, glycolysis, glycolysis capacity and reserve. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs siControl.

图5 PSME2和STAT3双重抑制相关差异表达基因和关键基因鉴定及Hallmark/KEGG富集分析

Fig.5 Identification of 45 differentially expressed genes (DEGs) and 9 key genes associated with dual inhibition of PSME2 and STAT3 and their Hallmark/KEGG enrichment analyses. A: Venn diagram showing 45 overlapping DEGs between PR171+WP1066 and siPSME2+WP1066 groups. B: Hallmark enrichment analysis of the overlapping DEGs. C: KEGG pathway enrichment of common DEGs. D: Intersection analysis of DEGs among PR171+WP1066, siPSME2+WP1066, siPSME2, WP1066, and PR171 groups. Nine candidate DEGs related to combined treatment are retained (Red indicates upregulation and blue indicates downregulation; log2FC > 0 upregulated genes, log2FC < 0 downregulated genes).

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PSME2和STAT3双重抑制通过调控SLC7A11/GPX4轴诱导食管鳞癌细胞铁死亡

Dual inhibition of PSME2 and STAT3 induces ferroptosis in esophageal squamous cell carcinoma cells by regulating the SLC7A11/GPX4 axis

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