FOXM1上调ELK1介导铁死亡促进宫颈癌细胞顺铂耐药性

FOXM1 mediates ferroptosis and promotes cisplatin resistance in cervical cancer cells by up-regulating ELK1

目的探讨FOXM1通过ELK1对宫颈癌细胞顺铂(DDP)耐药性的影响及其潜在机制。方法生物信息学分析FOXM1和ELK1在宫颈癌组织中的表达;qPCR检测FOXM1和ELK1在宫颈癌细胞中的表达;双荧光素酶报告基因实验、ChIP实验验证二者结合情况。GSEA分析ELK1富集通路;Pearson相关性分析ELK1和抑制铁死亡关键基因的相关性。CCK-8检测细胞活力;PI染色及流式细胞术检测细胞凋亡情况。透射电镜观察线粒体形态变化;Lipid Peroxidation(MDA)Assay试剂盒检测丙二醛(MDA)含量;Werstern blot检测GPX4、SLC7A11、ACSL4蛋白表达。结果ELK1与FOXM1在宫颈癌组织和细胞中显著高表达,且二者存在结合位点。与oe-NC组相比,oe-ELK1组细胞存活率和IC50值显著提高;而与si-NC组相比,si-ELK1组细胞存活率和IC50值显著降低。与oe-NC+DMSO组相比,oe-ELK1+DMSO组线粒体没有出现萎缩、线粒体脊减少情况;而与oe-ELK1+DMSO组相比,oe-ELK1+erastin组线粒体出现了明显的萎缩、线粒体脊减少甚至消失现象。与oe-NC+DMSO组相比,oe-ELK1+DMSO组细胞内MDA含量显著降低,GPX4、SLC7A11蛋白表达增加,ACSL4蛋白表达降低,进一步用erastin处理则逆转了这一现象。与oe-NC+DMSO组相比,oe-ELK1+DMSO组CaSki/DDP细胞的IC50值显著提高,而erastin处理能够减弱这一促进作用。此外,与si-NC+oe-NC组相比,si-FOXM1+oe-NC组线粒体出现了明显的萎缩、线粒体脊减少甚至消失现象;而与si-FOXM1+oe-NC组相比,si-FOXM1+oe-ELK1组线粒体形态得到恢复。与si-NC+oe-NC组相比,si-FOXM1+oe-NC组细胞内MDA含量显著提高,GPX4、SLC7A11蛋白表达显著降低,ACSL4蛋白表达显著提高;而与si-FOXM1+oe-NC组相比,si-FOXM1+oe-ELK1组细胞内MDA含量显著降低,GPX4、SLC7A11蛋白表达显著提高,ACSL4蛋白表达显著降低。与si-NC+oe-NC组相比,si-FOXM1+oe-NC转染抑制了不同浓度DDP处理CaSki/DDP细胞的IC50值,而同时oe-ELK1转染能够逆转这一抑制作用。结论FOXM1通过上调ELK1的表达介导铁死亡,进而促进宫颈癌细胞DDP耐药。

Objective To investigate the effect and potential mechanism of FOXM1 on cisplatin(DDP)resistance in cervical cancer cells through ELK1.Methods The expressions of FOXM1 and ELK1 in cervical cancer tissues were analyzed by bioinformatics.The expressions of FOXM1 and ELK1 in cervical cancer cells were detected by qPCR.Double luciferase reporter gene experiment and ChIP experiment were performed to verify the combination of the two.ELK1 enrichment pathway was analyzed by GSEA.The correlation between ELK1 and the key genes of inhibiting ferroptosis was analyzed by Pearson correlation analysis.Cell viability was detected by CCK-8,and cell apoptosis was detected by PI staining and flow cytometry.The morphological changes of mitochondria were observed by transmission electron microscopy.Lipid Peroxidation(MDA)Assay kit was used to detect the MDA content.The expressions of GPX4,SLC7A11 and ACSL4 were detected by Werstern blot.Results ELK1 and FOXM1 were highly expressed in cervical cancer tissues and cells,and and there were binding sites for them.Compared with the oe-NC group,the cell survival rate and IC50 value of the oe-ELK1 group were significantly increased.Compared with the si-NC group,the cell survival rate and IC50 value of the si-ELK1 group were significantly decreased.Compared with the oe-NC+DMSO group,mitochondrial atrophy and mitochondrial ridge reduction were not observed in the oe-ELK1+DMSO group.Compared with the oe-ELK1+DMSO group,it showed obvious mitochondriaatrophy,mitochondrial ridge reduction or even disappearance in the oe-ELK1+erastin group.Compared with the oe-NC+DMSO group,intracellular MDA content was significantly decreased,the expressions of GPX4 and SLC7A11 proteins were increased,and the expression of ACSL4 protein was decreased in the oe-ELK1+DMSO group.And the use of erastin had reversed the above phenomenon.Compared with the oe-NC+DMSO group,the IC50 value of CaSki/DDP cells in the oe-ELK1+DMSO group was significantly increased,while the use of erastin had reduced the facilitation.In addition,compared with the si-NC+oe-NC group,the si-FOXM1+oe-NC group showed obvious mitochondriaatrophy,mitochondrial ridge reduction or even disappearance.Compared with the si-FOXM1+oe-NC group,the mitochondrial morphology was restored in the si-FOXM1+oe-ELK1 group.Compared with the si-NC+oe-NC group,the intracellular MDA content in the si-FOXM1+oe-NC group was significantly increased,the expressions of GPX4 and SLC7A11 protein were decreased,and the expression of ACSL4 protein was increased.Compared with the si-FOXM1+oe-NC group,the intracellular MDA contentin the si-FOXM1+oe-ELK1 group was significantly decreased,and the expressions of GPX4 and SLC7A11 protein were increased,while the expression of ACSL4 protein was decreased.Compared with the si-NC+oe-NC group,the IC50 value of CaSki/DDP cells in the si-FOXM1+oe-NC group was significantly decreased.Meanwhile,oe-ELK1 transfection has reversed the inhibition effect.Conclusion FOXM1 can mediate ferroptosis by up-regulating the expression of ELK1,thus promoting DDP resistance in cervical cancer cells.