铁死亡诱导剂Erastin下调ACSL4抑制肝癌细胞体外增殖

doi:10.12122/j.issn.1673-4254.2024.11.09

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (11): 2131-2136.doi: 10.12122/j.issn.1673-4254.2024.11.09

• • 上一篇

铁死亡诱导剂Erastin下调ACSL4抑制肝癌细胞体外增殖

赵培培¹^,²(), 周志刚³(), 杨媛媛¹^,², 黄树升¹^,², 涂逸轩⁴, 涂剑¹^,²()

^1.桂林医学院药学院，广西桂林 541199
^2.广西肝脏损伤与修复分子医学重点实验室，广西桂林 541199
^3.桂林医学院第二附属医院麻醉科，广西桂林 541199
^4.暨南大学附属第一医院风湿免疫科，广东广州 510632

收稿日期:2024-08-22 出版日期:2024-11-20 发布日期:2024-11-29
通讯作者: 涂剑 E-mail:3180909244@qq.com;zhouzhigang0734@sina.com;tujian0734@aliyun.com
作者简介:赵培培，在读硕士研究生，E-mail: 3180909244@qq.com
周志刚，硕士，副教授，E-mail: zhouzhigang0734@sina.com
第一联系人：赵培培、周志刚共同为第一作者
基金资助:
国家自然科学基金(82060662);广西区自然科学基金(2022JJA140776)

Ferroptosis inducer Erastin inhibits proliferation of liver cancer cells in vitro by down-regulating ACSL4

Peipei ZHAO¹^,²(), Zhigang ZHOU³(), Yuanyuan YANG¹^,², Shusheng HUANG¹^,², Yixuan TU⁴, Jian TU¹^,²()

^1.College of Pharmacy, Guilin Medical University, Guilin 541199, China
^2.Guangxi Key Laboratory of Molecular Medicine for Liver Injury and Repair, Guilin 541199, China
^3.Department of Anesthesiology, Second Affiliated Hospital of Guilin Medical University, Guilin 541199, China
^4.Department of Rheumatology and Immunology, The First Affiliated Hospital of Jinan University, Guangzhou 510632, China

Received:2024-08-22 Online:2024-11-20 Published:2024-11-29
Contact: Jian TU E-mail:3180909244@qq.com;zhouzhigang0734@sina.com;tujian0734@aliyun.com
Supported by:
National Natural Science Foundation of China(82060662)

摘要/Abstract

摘要：

目的分析酯酰辅酶A合成酶长链家族成员4（ACSL4）在肝癌中的表达，探究铁死亡调控ACSL4对癌细胞增殖能力的影响。方法收集肝癌和癌旁正常肝组织临床样本，HE染色病理学鉴定后，微量法检测丙二醛（MDA）含量，RT-qPCR检测ACSL4与增殖细胞核抗原（PCNA）的mRNA表达，Western blotting检测ACSL4与PCNA的蛋白表达。体外培养Huh-7人肝癌细胞，先分为3组：即铁死亡诱导剂Erastin组、抑制剂 Fer-1组、以及Erastin 与Fer-1联合作用组；其中Erastin或Fer-1组均包含0、20、40、60、80、100 μmol/L共6个浓度，然后采用筛选的Erastin 浓度80 μmol/L+（0、30、60、90 μmol/L）Fer-1，筛选Fer-1浓度后再分为3组：对照组、80 μmol/L Erastin单独处理组、80 μmol/L Erastin+60 μmol/L Fer-1联合处理组，均作用48 h。干预ACSL4、PCNA的表达后，平板克隆实验检测细胞增殖能力的改变，微量法检测MDA含量的变化。结果相较于癌旁正常肝组织，肝癌组织中MDA含量降低（P<0.01），ACSL4、PCNA的mRNA和蛋白表达均显著增强（P<0.05）；Erastin可抑制ACSL4、PCNA的mRNA和蛋白表达（P<0.01），并抑制细胞增殖（P<0.001）、上调MDA含量（P<0.01）；单独使用Fer-1对细胞存活率无影响；但加入Erastin后再应用Fer-1，则可逆转Erastin对ACSL4、PCNA表达（P<0.05），细胞增殖能力的抑制（P<0.001），MDA含量的上调（P<0.05）。结论 ACSL4在肝癌中表达增强，Erastin可提高MDA含量、下调ACSL4表达，诱导肝癌细胞铁死亡，抑制癌细胞增殖；Fer-1则可逆转Erastin的上述作用。

关键词: 肝癌, 细胞增殖, Erastin, ACSL4, 铁死亡

Abstract:

Objective To investigate the expression of Acyl-CoA synthetase long-chain family member 4 (ACSL4) in liver cancer and its role in regulating ferroptosis and proliferation of liver cancer cells. Methods Clinical samples of liver cancer and adjacent normal liver tissues were examined for malondialdehyde (MDA) contents and for expressions of mRNA and protein expressions of ACSL4 and proliferating cell nuclear antigen (PCNA) using RT-qPCR and Western blotting. Human liver cancer Huh-7 cells were treated with Erastin (a ferroptosis inducer), Fer-1 (a ferroptosis inhibitor), or both, and the changes in expression levels of MDA, ACSL4 and PCNA were detected, and the cell proliferation was assessed with plate cloning assay. Results MDA contents were lower and ACSL4 and PCNA expressions were higher significantly in liver cancer tissues than in adjacent liver tissues. In Huh-7 cells, Erastin treatment significantly inhibited mRNA and protein expressions of ACSL4 and PCNA, suppressed cell proliferation, and increased MDA contents. Fer-1 alone did not produce significant effect on cell viability but reversed the effect of Erastin on ACSL4 and PCNA expressions, cell proliferation and MDA contents. Conclusion ACSL4 level is significantly overexpressed in liver cancer. Erastin increases MDA contents and down-regulates ACSL4 expression, thereby promoting ferroptosis and inhibiting proliferation of liver cancer cells, and these effects can be reversed by Fer-1.

Key words: liver cancer, cell proliferation, Erastin, Acyl-CoA synthetase long-chain family member 4, ferroptosis

赵培培, 周志刚, 杨媛媛, 黄树升, 涂逸轩, 涂剑. 铁死亡诱导剂Erastin下调ACSL4抑制肝癌细胞体外增殖[J]. 南方医科大学学报, 2024, 44(11): 2131-2136.

Peipei ZHAO, Zhigang ZHOU, Yuanyuan YANG, Shusheng HUANG, Yixuan TU, Jian TU. Ferroptosis inducer Erastin inhibits proliferation of liver cancer cells in vitro by down-regulating ACSL4[J]. Journal of Southern Medical University, 2024, 44(11): 2131-2136.

图/表 5

图1 临床肝组织HE染色及MDA含量检测

Fig.1 HE staining and MDA contents detection in liver cancer and adjacent tissues. A: HE staining (Original magnification: ×40). B: MDA contents in the tissues (n=6). **P<0.01 vs Normal liver tissues.

图2 临床肝组织中ACSL4、PCNA的mRNA和蛋白表达

图3 铁死亡调控对Huh-7细胞活力的影响

Fig.3 Effects of Erastin and Fer-1 on viability of Huh-7 cells assessed using CCK-8 assay. A: Effects of different concentrations of Erastin on viability of Huh-7 cells (*P<0.05, **P<0.01, ****P <0.0001 vs 0 µmol/L). B: Effects of different concentrations of Fer-1 on viability of Huh-7 cells. C: Effects of Erastin combined with Fer-1 on viability of Huh-7 cells (**P<0.01, ****P<0.0001 vs Control; ##P<0.01 vs 0 µmol/L Fer-1).

图4 铁死亡调控ACSL4对Huh-7细胞MDA含量及PCNA表达的影响

Fig. 4 Effects of Erastin and Fer-1 on MDA contents and ACSL4 and PCNA expressions in Huh-7 cells. A: Effects of Erastin and Fer-1 on MDA contents in Huh-7 cells. B: Effects of Erastin and Fer-1 on mRNA expressions of ACSL4 and PCNA (RT-qPCR). C: Effects of Erastin and Fer-1 on protein expressions of ACSL4 and PCNA (Western blotting). D: Relative protein expression levels of ACSL4 and PCNA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Control; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 vs Erastin.

图5 铁死亡调控ACSL4对Huh-7细胞增殖能力的影响

Fig.5 Effects of Erastin and Fer-1 on proliferation ability of Huh-7 cells. A: Plate cloning assay of the cells treated with Erastin and Fer-1. B: Number of cell clones. ***P<0.001 vs Control; ###P<0.001 vs Erastin.

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铁死亡诱导剂Erastin下调ACSL4抑制肝癌细胞体外增殖

Ferroptosis inducer Erastin inhibits proliferation of liver cancer cells in vitro by down-regulating ACSL4

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