GPX4表达促进铁死亡参与子宫内膜癌恶性行为的机制

Study on the Mechanism by Which GPX4 Expression Promotes Ferroptosis and Participates in Malignant Behavior of Endometrial Carcinoma

摘要

目的 探讨谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）表达促进铁死亡参与子宫内膜癌（endometrial carcinoma，EC）恶性行为的机制。方法 通过定量实时聚合酶链反应（quantitative polymerase chain reaction，qPCR）、蛋白印迹和免疫组化（immunohistochemistry，IHC）测定EC组织中GPX4的表达。培养KLE细胞，分为shNC组（n = 6）和shGPX4组（n = 6），Transwell、克隆形成测定和流式细胞研究GPX4对EC细胞增殖、迁移、细胞凋亡的影响。检测细胞内Fe²⁺、活性氧（reactive oxygen Species，ROS）和丙二醛（malondialdehyde，MDA）水平，JC染色检测线粒体膜电位（mitochondrial membrane Potential，MMP），GPX4酶活性试剂盒检测酶活性，ELISA法检测脂质过氧化物4-羟基壬烯醛（4-Hydroxynonenal，4-HNE）含量，蛋白印迹检测铁死亡关键蛋白酰基辅酶A合成酶长链家族成员4（acyl-coa synthetase long chain family member 4，ACSL4）和溶质载体家族7成员11（solute Carrier family 7 member 11，SLC7A11）的表达；建立裸鼠EC异种移植肿瘤模型，分为shNC组（n = 6）和shGPX4组（n = 6），通过检测其重量体积、重量、Fe²⁺和MDA水平评价GPX4敲低在体内的效果。结果 GPX4在EC组织及细胞系中高表达[EC组织GPX4 mRNA：（118.1±6.92）vs 癌旁（62.72±5.20），t = 15.68，P < 0.001，Cohen's d=8.62]；与shNC组相比，shGPX4组KLE细胞增殖（克隆数：0.37±0.05 vs 0.89±0.07，t = 14.94，P < 0.001）、迁移能力显著降低，凋亡率升高[（61.64±7.03）% vs （12.60±2.48）%，t = 16.12，P < 0.001）]，细胞内Fe²⁺、脂质ROS、MDA、4-HNE水平升高，GSH及GPX4酶活性降低，MMP破坏，ACSL4表达上调、SLC7A11下调（P < 0.001）；裸鼠模型中，shGPX4组肿瘤体积[（0.36±0.07）vs （0.87±0.12）cm³，t = 9.07，P < 0.001]、重量显著减小，肿瘤组织铁含量及MDA水平升高（P < 0.001）。结论 研究表明GPX4沉默能诱导铁死亡的发生，进而降低子宫内膜癌细胞的增殖和侵袭。

Abstract

Objective To investigate the mechanism by which Glutathione Peroxidase 4 (GPX4) expression promotes ferroptosis and participates in the malignant behavior of Endometrial Carcinoma (EC). Methods The expression of GPX4 in EC tissues was determined by quantitative real-time PCR (qPCR), Western blot, and immunohistochemistry (IHC). KLE cells were cultured and divided into an shNC group (n = 6) and an shGPX4 group (n = 6). The effects of GPX4 on EC cell proliferation, migration, and apoptosis were investigated using Transwell assay, colony formation assay, and flow cytometry. Intracellular Fe²⁺, reactive oxygen species (ROS), and malondialdehyde (MDA) levels were detected. Mitochondrial membrane potential (MMP) was measured by JC-1 staining. GPX4 enzyme activity was assessed with a commercial kit. The content of lipid peroxide 4-hydroxynonenal (4-HNE) was determined by ELISA; and the expression of key ferroptosis-related proteins, Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) and Solute Carrier Family 7 Member 11 (SLC7A11), was analyzed by Western blot. An EC xenograft tumor model in nude mice was established and divided into an shNC group (n = 6) and an shGPX4 group (n = 6). The in vivo effect of GPX4 knockdown was evaluated by measuring tumor volume, weight, and levels of Fe²⁺ and MDA. Results GPX4 was highly expressed in EC tissues and cell lines (GPX4 mRNA in EC tissues: 118.1±6.92 vs. 62.72±5.20 in adjacent normal tissues, t = 15.68, P < 0.001, Cohen's d=8.62). Compared with the shNC group, the shGPX4 group showed significantly decreased KLE cell proliferation (colony number: 0.37±0.05 vs. 0.89±0.07, t = 14.94, P < 0.001) and migration capacity, along with an increased apoptosis rate (61.64±7.03% vs. 12.60±2.48%, t = 16.12, P < 0.001). Additionally, in the shGPX4 group, intracellular levels of Fe²⁺, lipid ROS, MDA, and 4-HNE were increased; GSH level and GPX4 enzyme activity were decreased; MMP was impaired; and the expression of ACSL4 was up-regulated while that of SLC7A11 was down-regulated (all P < 0.001). In the nude mouse model, the shGPX4 group showed significantly reduced tumor volume (0.36±0.07 vs. 0.87±0.12 cm³, t = 9.07, P < 0.001) and weight, along with increased iron content and MDA levels in tumor tissues (both P < 0.001). Conclusion The study demonstrates that GPX4 silencing can significantly induce ferroptosis, thereby reducing the proliferation and invasion of endometrial cancer cells.

关键词

GPX4 / 铁死亡 / 子宫内膜癌 / 恶性行为

Key words

GPX4 / Ferroptosis / Endometrial cancer / Vicious behavior

参考文献

[1] Crosbie E J, Kitson S J, McAlpine J N, et al. Endometrial cancer[J]. Lancet, 2022, 399(10333): 1412-1428.

[2] Nees L K, Heublein S, Steinmacher S, et al. Endometrial hyperplasia as a risk factor of endometrial cancer[J]. Arch Gynecol Obstet, 2022, 306(2): 407-421.

[3] Bogani G, Monk B J, Powell M A, et al. Adding immunotherapy to first-line treatment of advanced and metastatic endometrial cancer[J]. Ann Oncol, 2024, 35(5): 414-428.

[4] Tronconi F, Nero C, Giudice E, et al. Advanced and recurrent endometrial cancer: State of the art and future perspectives[J]. Crit Rev Oncol Hematol, 2022, 180: 103851.

[5] Liu J, Kang R, Tang D. Signaling pathways and defense mechanisms of ferroptosis[J]. Febs J, 2022, 289(22): 7038-7050.

[6] Li P, Jiang M, Li K, et al. Glutathione peroxidase 4-regulated neutrophil ferroptosis induces systemic autoimmunity[J]. Nat Immunol, 2021, 22(9): 1107-1117.

[7] Xie Y, Kang R, Klionsky D J, et al. GPX4 in cell death, autophagy, and disease[J]. Autophagy, 2023, 19(10): 2621-2638.

[8] Sekhar K R, Hanna D N, Cyr S, et al. Glutathione peroxidase 4 inhibition induces ferroptosis and mTOR pathway suppression in thyroid cancer[J]. Sci Rep, 2022, 12(1): 19396.

[9] Xue Q, Yan D, Chen X, et al. Copper-dependent autophagic degradation of GPX4 drives ferroptosis[J]. Autophagy, 2023, 19(7): 1982-1996.

[10] Allred D C. Prognostic and predictive factors in breast cancer by immunohistochemical analysis[J]. Mod Pathol, 1998, 11(2): 155-168.

[11] Liang D, Feng Y, Zandkarimi F, et al. Ferroptosis surveillance independent of GPX4 and differentially regulated by sex hormones[J]. Cell, 2023, 186(13): 2748-2764.e22.

[12] Žalytė E. Ferroptosis, metabolic rewiring, and endometrial cancer[J]. Int J Mol Sci, 2023, 25(1): 75.

[13] 陈茜, 陈梅. 过表达SALL4促进人宫颈癌细胞系的迁移和侵袭[J]. 河北医科大学学报, 2025, 46(10): 1160-1167.

[14] 王金红, 郝丹丹, 王玉敏. 非编码RNA调控铁死亡介导肺癌耐药研究进展[J]. 河北医科大学学报, 2025, 46(7): 792-795.

[15] Cheng L, He Q, Liu B, et al. SGK2 promotes prostate cancer metastasis by inhibiting ferroptosis via upregulating GPX4[J]. Cell Death Dis, 2023, 14(1): 74.

[16] 虞晶晶, 穆丹, 朱庆丰. 铁死亡研究进展与恶性肿瘤治疗[J]. 河北医科大学学报, 2022, 43(3): 363-367.

[17] Liu Y, Wan Y, Jiang Y, et al. GPX4: The hub of lipid oxidation, ferroptosis, disease and treatment[J]. Biochim Biophys Acta BBA Rev Cancer, 2023, 1878(3): 188890.

[18] Wang Z, Shu W, Zhao R, et al. Sodium butyrate induces ferroptosis in endometrial cancer cells via the RBM3/SLC7A11 axis[J]. Apoptosis, 2023, 28(7-8): 1168-1183.

[19] 刘茜, 曹钰洁, 李建英. 铁死亡在肺纤维化中作用机制的研究进展[J]. 河北医科大学学报, 2024, 45(4): 413-417.

基金资助

河北省医学科学研究基金(20241583)