[脆性X智力低下蛋白的高表达通过激活RAS/MAPK信号通路抑制结直肠肿瘤细胞的铁死亡]

[High expression of fragile X mental retardation protein inhibits ferroptosis of colorectal tumor cells by activating the RAS/MAPK signaling pathway].

作者信息

Wang N, Shi B, Man X, Wu W, Cao J

机构信息

Master's Cultivation Station, Ningxia Medical University General Hospital, Yinchuan 750004, China.

Department of Emergency Medicine, Ningxia Medical University General Hospital, Yinchuan 750004, China.

出版信息

Nan Fang Yi Ke Da Xue Xue Bao. 2024 May 20;44(5):885-893. doi: 10.12122/j.issn.1673-4254.2024.05.10.

DOI:10.12122/j.issn.1673-4254.2024.05.10

PMID:38862446

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11166710/

Abstract

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/Fe 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, Fe 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.

摘要

目的

探讨脆性X智力低下蛋白（FMRP）调节结直肠癌（CRC）细胞铁死亡逃逸的机制。

方法

我们使用RT-qPCR和蛋白质印迹法检测CRC细胞系中FMRP的表达水平，并使用TCGA数据库分析FMRP介导的CRC进展调节中涉及的生物学功能和信号通路。构建慢病毒FMRP过表达载体（Lv-FMRP）和3种敲低载体（siFMRP-1、siFMRP-2和siFMRP-3），使用CCK8法和平板克隆形成试验检测它们对HCT116细胞增殖的影响；使用MDA/ROS/GSH/Fe试剂盒测定细胞铁死亡水平的变化，使用JC-1荧光染色检测线粒体膜电位变化，使用蛋白质印迹法检测与铁死亡和RAS/MAPK信号通路相关的蛋白质表达。在裸鼠中评估转染细胞的皮下致瘤潜力。

结果

与正常结肠黏膜上皮NCM460细胞相比，CRC细胞系中FMRP表达水平显著更高。生物信息学分析表明FMRP参与调节活性氧、氧化应激诱导的细胞死亡、线粒体呼吸和谷胱甘肽代谢途径。在细胞实验中，FMRP敲低显著抑制HCT116细胞增殖，降低细胞内谷胱甘肽（GSH）含量，增加丙二醛（MDA）和活性氧（ROS）水平、铁荧光强度和线粒体膜电位，降低溶质载体家族7成员11（SLC7A11）/谷胱甘肽过氧化物酶4（GPX4）蛋白表达以及细胞外信号调节激酶（ERK）、丝裂原活化蛋白激酶激酶（MEK）、丝裂原活化蛋白激酶（MAPK）和RAS蛋白的磷酸化水平；FMRP过表达导致细胞出现相反的变化。在荷瘤裸鼠中，FMRP敲低的HCT116细胞致瘤潜力减弱，异种移植瘤生长速率降低，异种移植瘤中SLC7A11表达减少。