摘要
目的:探讨miR-324-3p对前列腺癌(PCa)细胞铁死亡的影响及其分子作用机制。方法:通过qRT-PCR检测PCa组织、癌旁组织和细胞系中miR-324-3p和GPX4 mRNA的表达水平;采用Western blot检测PCa细胞系中谷胱甘肽过氧化物酶4(GPX4)蛋白的表达水平;CCK-8检测PCa细胞增殖活力;运用谷胱甘肽(GSH)试剂盒检测PCa细胞中GSH水平,脂质过氧化(MDA)试剂盒检测PCa细胞脂质氧化水平,DCFH-DA荧光探针法检测PCa细胞中活性氧(ROS)水平;双萤光素酶报告基因验证miR-324-3p和GPX4的靶向关系。结果:PCa组织中miR-324-3p的表达水平低于对应的癌旁组织,且PCa细胞中miR-324-3p的表达水平低于正常前列腺上皮细胞;而PCa组织和细胞中GPX4的表达水平高于对应的癌旁组织和正常前列腺上皮细胞。双萤光素酶报告实验表明,miR-324-3p直接靶向负调控GPX4。过表达miR-324-3p可明显抑制PCa细胞增殖活力,降低了GSH水平,上调了脂质氧化水平和ROS水平;而铁死亡抑制剂Fer-1或GPX4可逆转miR-324-3p对PCa细胞铁死亡的促进作用。结论:miR-324-3p通过靶向负调控GPX4的表达促进PCa细胞铁死亡,进而在PCa发挥抗癌作用。
Objective:To explore the effects of miR-324-3 p on prostate cancer(PCa)cell ferroptosis and its underlying mechanism.Methods:qRT-PCR was performed to detect the expression of miR-324-3 p and glutathione peroxidase 4(GPX4)mRNA in PCa tissues,matched adjacent tissues,and cell lines.Western blot was employed to examine the protein level of GPX4 in PCa cell lines and normal prostate epithelial cells.CCK-8 assay was used to evaluate cell proliferation.The levels of glutathione(GSH),lipid oxidation,and reactive oxygen species(ROS)were determined using GSH detection assay kit,lipid peroxidation MDA assay kit and DCFH-DA fluorescent probe assay.Dual-luciferase reporter gene was applied to verify the interaction of miR-324-3 p and GPX4.Results:The expression of miR-324-3 p in PCa tissues was lower than that in the corresponding paracancerous tissues,and the expression of miR-324-3 p was downregulated in PCa cell lines compared with normal prostate epithelial cells.GPX4 was highly-expressed in PCa tissues and cell lines in comparison with the corresponding paracancerous tissues and normal prostate epithelial cells.Dual-luciferase reporter results showed that miR-324-3 p was directly targeted to negatively regulate GPX4.Overexpression of miR-324-3 p significantly inhibited PCa cell proliferation,reduced GSH production and enhanced the levels of lipid oxidation and ROS;while treatment with the ferroptosis inhibitor Fer-1 or GPX4 reversed the promotion effect of miR-324-3 p on ferroptosis in PCa cells.Conclusion:miR-324-3 p promoted ferroptosis in PCa cells by targeting negative regulation of GPX4 expression and thus exerted anticancer effects on PCa.
作者
张赛
叶纪伟
沈远径
穆克飞
郭新武
ZHANG Sai;YE Ji-wei;SHEN Yuan-jing;MU Ke-fei;GUO Xin-wu(Nanyang Second General Hospital,Nanyang 473000,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2022年第1期72-79,共8页
China Biotechnology
基金
南阳市科技发展计划(KJGG2018124)资助项目
