Study on the Mechanism by Which GPX4 Expression Promotes Ferroptosis and Participates in Malignant Behavior of Endometrial Carcinoma
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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. A 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. -
Key words:
- GPX4 /
- Ferroptosis /
- Endometrial cancer /
- Vicious behavior
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图 1 GPX4 在 EC 组织中高表达
A：蛋白印迹检测 GPX4 蛋白的表达；B：GPX4的统计学分析；C：GPX4的免疫组织化典型图片（×200）；D：GPX4表达量统计分析；*P < 0.05。
Figure 1. GPX4 is highly expressed in EC tissues
图 2 GPX4在子宫内膜上皮细胞及不同子宫内膜癌细胞系中的表达
A：蛋白印迹检测ACSL4 表达和SLC7A11表达；B：ACSL4统计学分析；C：SLC7A11表达；D：蛋白印迹分析 GPX4 蛋白的表达；E：qPCR检测GPX4表达。
Figure 2. Expression of GPX4 in endometrial epithelial cells and different endometrial cancer cell lines
图 3 GPX4对EC细胞增殖和迁移的影响（×400）
A：集落形成试验检测细胞增殖；B： transwell 试验测定细胞迁移能力；C：流式细胞仪分析细胞凋亡。
Figure 3. Effects of GPX4 on the proliferation and migration of EC cells （×400）
图 4 GPX4 敲低诱导 EC 细胞铁死亡（×400）
JC-1染色检测MMP。
Figure 4. GPX4 knockdown induces ferroptosis in EC cells （×400）
图 5 GPX4 敲低对 EC 细胞脂质过氧化物及铁死亡关键蛋白的影响
A：蛋白印迹检测ACSL4 表达和SLC7A11表达；B：ACSL4的统计学分析；C：SLC7A11的统计学分析；*P < 0.05。
Figure 5. Effects of GPX4 knockdown on lipid peroxides and key ferroptosis proteins in EC Cells
图 6 GPX4 敲低增强体内铁死亡活性
A：BALB/c 裸鼠的异种移植肿瘤图像；B：异种移植肿瘤中 GPX4 表达的免疫组织化学和 HE 染色（×400）。
Figure 6. GPX4 knockdown enhances ferroptosis activity in vivo
表 1 引物序列
Table 1. Primer sequence
基因名称 引物方向 引物序列 （5' → 3'） GPX4 上游 Forward GAGGCAAGACCGAAGAGAAACTAC 下游 Reverse CCGAACTGGTTACACGGGAA GAPDH 上游 Forward GAGTCAACGGATTTGGTCGT 下游 Reverse GACAAGCTTCCCGTTCTCAG 
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表 2 GPX4在子宫内膜上皮细胞及不同子宫内膜癌细胞系中的表达（$\bar x \pm s $）
Table 2. Expression of GPX4 in endometrial epithelial cells and different endometrial cancer cell lines （$\bar x \pm s $）
分组 GPX4 mRNA EECs 1.09 ± 0.07 Ishikawa 5.05 ± 0.59* RL95-2 6.54 ± 0.61* HEC1A 6.98 ± 1.32* HEC1B 7.59 ± 1.32* KLE 8.41 ± 1.34* F 32.72 P <0.001* η2 0.84 多组比较，One-way ANOVA，事后检验Tukey HSD，*P < 0.05。
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表 3 GPX4对EC细胞增殖和迁移的影响（$\bar x \pm s $）
Table 3. Effects of GPX4 on EC cell proliferation and migration （$\bar x \pm s $）
项目 shNC组（n=6） shGPX4组（n=6） t P df Cohen’s d 克隆的相对（折叠）数 0.89 ± 0.07 0.37 ± 0.05 14.94 <0.001* 10 8.21 细胞的相对（折叠）数 0.99 ± 0.07 0.33 ± 0.06 16.72 <0.001* 10 9.24 细胞凋亡率（%） 12.60 ± 2.48 61.64 ± 7.03 16.12 <0.001* 10 8.91 独立样本t检验，*P < 0.05。
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表 4 GPX4 敲低诱导 EC 细胞铁死亡（$\bar x \pm s $）
Table 4. GPX4 knockdown induces ferroptosis in EC cells （$\bar x \pm s $）
项目 shNC组（n=6） shGPX4组（n=6） t P df Cohen’s d 细胞内亚铁水平（uM/g） 9.77 ± 1.04 17.46 ± 1.64 9.68 <0.001* 10 5.34 脂质 ROS 水平 1.33 ± 0.11 2.04 ± 0.19 8.15 <0.001* 10 4.51 MDA 水平（nmol/mg） 3.33 ± 1.03 8.17 ± 1.17 7.59 <0.001* 10 4.19 独立样本t检验，*P < 0.05。
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表 5 GPX4 敲低对 EC 细胞脂质过氧化物及铁死亡关键蛋白的影响（$\bar x \pm s $）
Table 5. Effects of GPX4 knockdown on lipid peroxide and expression of key ferroptosis proteins in EC cells （$\bar x \pm s $）
项目 shNC组（n=6） shGPX4组（n=6） t P df Cohen’s d 细胞内 GSH 水平（nmol/mg prot） 28.64 ± 3.12 12.37 ± 2.05 11.82 <0.001* 10 6.53 GPX4 酶活性（U/mg prot） 15.26 ± 1.89 5.73 ± 1.08 10.95 <0.001* 10 6.06 4-HNE（ng/mL） 18.25 ± 2.31 56.78 ± 4.92 16.03 <0.001* 10 8.86 独立样本t检验，*P < 0.05。
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表 6 GPX4 敲低增强体内铁死亡活性（$\bar x \pm s $）
Table 6. GPX4 knockdown enhances ferroptosis activity in vivo （$\bar x \pm s $）
项目 shNC组（n=6） shGPX4组（n=6） t P df Cohen’s d 肿瘤体积（cm3） 0.87 ± 0.12 0.36 ± 0.07 9.07 <0.001* 10 5.01 肿瘤重量（g） 0.73 ± 0.07 0.32 ± 0.06 10.53 <0.001* 10 5.81 细胞铁含量（μmol/g prot） 4.02 ± 0.80 7.89 ± 1.75 4.94 <0.001* 10 2.73 MDA 水平（nmol/mg） 23.35 ± 3.71 42.02 ± 5.78 6.67 <0.001* 10 3.68 独立样本t检验，*P < 0.05。
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