JNK通路介导的铁死亡在三阴性乳腺癌细胞增殖及凋亡中的作用

doi:10.3969/j.issn.1004-616x.2023.05.002

摘要/Abstract

摘要： 目的： 研究c-Jun 氨基末端激酶(JNK)通路介导的铁死亡在三阴性乳腺癌(TNBC)细胞增殖及凋亡中的作用。方法： 收集2020年1月—2022年5月佳木斯大学附属第一医院肿瘤科手术切除的105例TNBC癌组织和癌旁正常组织，并分别培养TNBC细胞系 MDA-MB-231 以及非 TNBC 细胞系 MCF-7、SK-BR-3，采用 Western blot 检测 TNBC 组织、癌旁正常组织、TNBC 细胞系、非TNBC 细胞中磷酸化 JNK(p-JNK)、铁死亡标志基因转铁蛋白受体 1(TFR1)和谷胱甘肽过氧化物酶 4(GPX4)的表达水平。另将MDA-MB-231细胞分为对照组、JNK激动剂组(5 μmol/L ANISO)、铁死亡抑制剂组(2 μmol/L Ferrostatin-1)、激动剂+抑制剂组(5μmol/L ANISO+2 μmol/L Ferrostatin-1)。各组细胞分别处理24 h后，采用CCK-8法检测细胞增殖抑制率，TUNEL法检测细胞凋亡率，分别采用试剂盒检测谷胱甘肽过氧化物酶(GSH)、超氧化物歧化酶(SOD)的活性及丙二醛(MDA)的含量；采用荧光定量PCR和Western blot检测细胞中TFR1、GPX4 mRNA和蛋白的表达水平。结果： 与癌旁组织比较，TNBC组织中p-JNK、TFR1的表达水平下降(P<0.05)，GPX4的表达水平升高(P<0.05)，且p-JNK与TFR1表达水平呈正相关(r=0.515，P<0.05)，p-JNK与GPX表达水平呈负相关(r=-0.442，P<0.05)。与MCF-7、SK-BR-3细胞相比，MDA-MB-231细胞中p-JNK、TFR1表达水平降低(P<0.05)，而GPX4表达水平升高(P<0.05)。与对照组比较，JNK激动剂组细胞的增殖抑制率、凋亡率、TFR1表达水平、MDA含量均增加(P<0.05)，GPX4表达水平、GSH和SOD活性降低(P<0.05)；铁死亡抑制剂组的增殖抑制率、凋亡率、GSH和SOD活性、MDA含量均无显著变化(P>0.05)，但TFR1表达水平降低、GPX4表达水平增加(P<0.05)；与JNK激动剂组比较，激动剂+抑制剂组细胞的增殖抑制率、凋亡率、TFR1表达水平、MDA含量均降低(P<0.05)，而GPX4表达水平、GSH和SOD活性增加(P<0.05)。结论： JNK通路通过激活铁死亡的方式抑制TNBC细胞增殖，促进TNBC细胞凋亡。

关键词: 三阴性乳腺癌, c-Jun氨基末端激酶, 铁死亡, 增殖, 凋亡

Abstract: OBJECTIVE: To study the role of the c-Jun N-terminal kinase (JNK) pathway-mediated ferroptosis in the proliferation and apoptosis of triple negative breast cancer (TNBC). METHODS: From January 2020 to May 2022,105 cases of TNBC tissues and adjacent tissues which were resected surgically in the First Affiliated Hospital of Jiamusi University were collected, TNBC cell line MDA-MB-231 and non TNBC cell lines MCF-7, SK-BR-3 were cultured. Expression levels of p-JNK and ferroptosis marker gene transferrin receptor 1 (TFR1) and glutathione peroxidase 4 (GPX4) in the tissues and the TNBC cells and non TNBC cells were detected. MDA-MB-231 cells in cultures were divided into the control and the JNK agonist groups (5 μmol/L ANISO),the ferroptosis inhibitor (2 μmol/L ferrostatin-1),and the agonist+inhibitor groups (5 μmol/L ANISO+2 μmol/L ferrostatin-1). After 24h treatment in each group, proliferation inhibition rates were detected using the CCK-8 assay,apoptosis rates by the TUNEL assay,activities of glutathione peroxidase (GSH),superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were by kits,and expression levels of p-JNK, TFR1 and GPX4 were by PCR and western blot. RESULTS: Compared with adjacent tissues,expression levels of p-JNK and TFR1 in the TNBC tissues were decreased,but the levels for GPX4 were increased (P<0.05). p-JNK expressions were positively correlated with TFR1(correlation coefficient r=0.515,P<0.05) and negatively correlated with GPX4 (correlation coefficient r=-0.442,P<0.05). Compared with the MCF-7 and SK-BR-3 cells,expression levels of p-JNK and TFR1 in MDA-MB-231 were decreased,and expression levels of GPX4 were increased (P<0.05). Compared with the control group,proliferation inhibition rates,apoptosis rates,the expression levels of TFR4,and contents of MDA were increased (P<0.05),while expression levels of GPX4,and activities of GSH and SOD were decreased in the JNK agonist group (P<0.05). In addition,proliferation inhibition rates,apoptosis rates,activities of GSH and SOD,and contents of MDA showed no significant changes (P>0.05),while expression levels of TFR4 were decreased and expression levels of GPX4 were increased (P<0.05) in the ferroptosis inhibitor group. Compared with JNK agonist group, proliferation inhibition rates,apoptosis rates,expression levels of TFR4,contents of MDA were decreased (P<0.05), while expression levels of GPX4, and activities of GSH and SOD were increased in the agonist + inhibitor group (P<0.05). CONCLUSION: Activation of the JNK pathway inhibited proliferation and promoted apoptosis of TNBC cell by activating ferroptosis.

Key words: triple negative breast cancer, c?Jun N?terminal kinase, ferroptosis, proliferation, apoptosis

中图分类号:

R737.9

张晨欣, 周昱. JNK通路介导的铁死亡在三阴性乳腺癌细胞增殖及凋亡中的作用[J]. 癌变·畸变·突变, 2023, 35(5): 334-340.

ZHANG Chenxin, ZHOU Yu. Role of JNK pathway mediated ferroptosis in proliferation and apoptosis of triple negative breast cancer cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2023, 35(5): 334-340.

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