目的 探讨脆性X智力障碍蛋白(FMRP)调控结直肠肿瘤(CRC)细胞逃避铁死亡的作用机制.方法 使用RT-qPCR和Western blotting方法验证FMRP在CRC细胞中的表达;利用TCGA数据库分析FMRP参与调控CRC进展的生物功能及信号通路;采用慢病毒表达系统和siRNA干扰技术,分别构建FMRP过表达载体(Lv-FMRP)和敲低载体(siFMRP-1、siFMRP-2、siFMRP-3),细胞实验设Control组、NC组、siFMRP-1组、siFMRP-2组、siFMRP-3组、Lv-NC组、Lv-FMRP组;采用CCK8和平板克隆实验检测细胞增殖;采用MDA/ROS/GSH/Fe2+试剂盒检测细胞铁死亡水平;采用JC-1荧光染色法检测线粒体膜电位变化;采用Western blot检测铁死亡相关蛋白及RAS/MAPK信号通路相关蛋白表达;利用裸鼠皮下成瘤实验观察FMRP对肿瘤生长的影响.结果 与正常肠粘膜上皮细胞NCM460相比,FMRP在CRC细胞中明显高表达(P<0.05);TCGA数据库联合生信分析显示FMRP与活性氧调控、氧化应激诱导细胞死亡、线粒体呼吸等生物过程相关,与谷胱甘肽代谢通路相关;体外实验结果显示,与Control组相比,FMRP敲低组细胞增殖能力降低,GSH含量降低,MDA和ROS含量升高,Fe2+荧光强度增强(P<0.05),SLC7A11/GPX4蛋白表达降低,线粒体膜电位JC-1荧光强度升高,而FMRP过表达组与上述结果相反;体内实验结果显示,敲低FMRP抑制瘤体生长,抑制SLC7A11表达(P<0.01);进一步检测RAS/MAPK信号通路,发现敲低FMRP导致ERK、MEK、MAPK、RAS蛋白磷酸化水平降低,过表达FMRP上述蛋白磷酸化水平升高(P<0.05).结论 FMRP通过激活RAS/MAPK信号通路抑制细胞铁死亡促进CRC恶性进展.
Objective To investigate the mechanism by which fragile X mental retardation protein(FMRP)regulates ferroptosis evasion in colorectal cancer(CRC)cells.Methods We examined FMRP expression levels in CRC cell lines using RT-qPCR and Western blotting and analyzed the biological functions and signaling pathways involved in FMRP-mediated regulation of CRC progression using the TCGA database.A lentiviral FMRP overexpression vector(Lv-FMRP)and 3 knockdown vectors(siFMRP-1,siFMRP-2,and siFMRP-3)were constructed,and their effects on proliferation of HCT116 cells were examined using CCK8 assay and plate clone formation assay;the changes in cell ferroptosis level was determined using MDA/ROS/GSH/Fe2+kits,mitochondrial membrane potential changes were detected using JC-1 fluorescence staining,and the expressions of proteins associated with ferroptosis and the RAS/MAPK signaling pathway were detected using Western blotting.The subcutaneous tumorigenic potential of the transfected cells was evaluated in nude mice.Results Compared with normal colonic mucosal epithelial NCM460 cells,the CRC cell lines had significantly higher FMRP expression level.Bioinformatics analysis suggested the involvement of FMRP in regulation of reactive oxygen,oxidative stress-induced cell death,mitochondrial respiration,and glutathione metabolism pathways.In the cell experiments,FMRP knockdown significantly inhibited proliferation of HCT116 cells,lowered cellular GSH content,increased MDA and ROS levels,Fe2+fluorescence intensity,and mitochondrial membrane potential,and decreased SLC7A11/GPX4 protein expressions and the phosphorylation levels of ERK,MEK,MAPK,and RAS proteins;FMRP overexpression resulted in the opposite changes in the cells.In the tumor-bearing nude mice,HCT116 cells with FMRP knockdown showed attenuated tumorigenic potential with lowered xenograft growth rate and reduced SLC7A11 expression in the xenograft.Conclusion The high expression of FMRP inhibits ferroptosis in CRC cells and promotes progression of CRC by activating the RAS/MAPK signaling pathway.
王南;石斌;马小兰;吴伟超;曹佳
宁夏医科大学总医院 硕士培养站,宁夏 银川 750004宁夏医科大学总医院 急诊科,宁夏 银川 750004银川市第一人民医院耳鼻喉科,宁夏 银川 750004宁夏医科大学总医院 医学科学研究院外科学研究室,宁夏 银川 750004
FMRP;铁死亡;RAS/MAPK信号通路;结直肠肿瘤
fragile X mental retardation protein;ferroptosis;RAS/MAPK signaling pathway;colorectal cancer
《南方医科大学学报》 2024 (005)
885-893 / 9
宁夏回族自治区重点研发计划项目(2022CMG03124);宁夏回族自治区重点研发(引才专项)项目(2021BEB04046);宁夏回族自治区第六批自治区青年科技人才托举工程项目(NXKJTJ2021119)
10.12122/j.issn.1673-4254.2024.05.10
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