摘要
目的 探究铁死亡在肝癌细胞对仑伐替尼(Lenva)耐药中的作用,为预防或逆转Lenva耐药提供新思路.方法 体外培养人肝癌细胞系PLC/PRF/5,即亲本细胞(PC)采用梯度浓度诱导法构建Lenva耐药细胞(LRC);利用RNA-seq技术对LRC和PC两种细胞进行转录组测序,测序结果进行差异表达基因分析及KEGG富集分析;采用CCK-8 法测定细胞活力,采用免疫印迹检测铁死亡相关蛋白,利用DHE探针检测细胞活性氧(ROS),应用透射电镜观察细胞线粒体形态.结果 Lenva处理PC和LRC 24 h,半数抑制浓度(IC50)分别为24.88 μmol/L和89.34 μmol/L,提示Lenva耐药人肝癌细胞体外诱导成功.RNA-seq测序检测到12106 个基因,与PC比较,LRC 88 个基因上调,197 个基因表达下调.差异基因的 KEGG 通路富集分析结果显示,铁死亡信号通路位列其中.利用10 μmol/L Lenva处理细胞48 h,与PC相比,LRC铁死亡相关蛋白SLC7A11 和GPX4 表达增加,ROS含量降低.电镜结果显示,与PC相比,特征性铁死亡线粒体形态改变的LRC明显减少.结论 肝癌细胞对Lenva耐药可能涉及多种基因和信号途径,其中铁死亡抵抗机制可能是引起肝癌细胞耐Lenva的关键.
Abstract
Objective To explore the role of ferroptosis in the resistance of liver cancer cells to lenvatinib(Lenva),and provide new ideas for preventing or reversing Lenva resistance.Methods Human liver cancer cell line PLC/PRF/5 cells also known as parental cells(PC)were cultured in vitro,and Lenva resistant cells(LRG)were constructed using gradient concentration induction method.RNA-seq technology was used for transcriptome sequencing of LRC and PC.Differential expression gene analysis and KEGG enrichment analysis were completed.Cell viability was measured by CCK-8 assay.Expressions of ferroptosis related proteins were detected by Western blot.The reactive oxygen species(ROS)was detected using DHE probes,and changes in mitochondrial morphology were observed by transmission electron microscopy(TEM).Results The half inhibitory concentration IC50 of Lenva in PC group and LRC group was 24.88 μmol/L and 89.34 μmol/L,respectively,indicating the successful induction of Lenva resistant human liver cancer cells in vitro.A total of 12106 genes were detected by RNA-seq,among which 88 genes were upregulated and 197 genes were downregulated in the LRC group compared to the PC group.The KEGG pathway enrichment analysis of differential expression genes showed that ferroptosis signaling pathway was enriched within it.Under the exposure of Lenva(10 μmol/L for 48 h),the expression of ferroptosis related proteins SLC7A11 and GPX4 were increased in the LRC group compared to the PC group,while the content of ROS was decreased.TEM results showed that compared to the PC group,the characteristic mitochondrial morphological changes related to ferroptosis in the LRC group were significantly reduced.Conclusion Lenva resistance in liver cancer cells may involve multiple genes and signaling pathways,among which ferroptosis resistance may be a key role in causing Lenva resistance.
基金项目
国家自然科学基金项目(81960520)
广西科技厅中央引导地方科技发展资金项目(桂科ZY21195024)
广西自然科学基金重点项目(2020GXNSFDA238006)
桂林市第三批漓江学者支持计划(2022-5-07)
NETL
NSTL
万方数据