PDF(Pubmed)
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/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.
CONCLUSIONS: The high expression of FMRP inhibits ferroptosis in CRC cells and promotes progression of CRC by activating the RAS/MAPK signaling pathway.
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.
CONCLUSIONS: 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)调控结直肠癌(CRC)细胞铁凋亡逃逸的机制。
方法：我们使用RT-qPCR和Western印迹检测了CRC细胞系中FMRP的表达水平，并使用TCGA数据库分析了FMRP介导的CRC进展调控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个敲低载体(siFMRP-1、siFMRP-2和siFMRP-3),并使用CCK8法和平板克隆形成法检测其对HCT116细胞增殖的影响；使用MDA/ROS/GSH/Fe2试剂盒测定细胞铁凋亡水平的变化，使用JC-1荧光染色检测线粒体膜电位变化，免疫印迹法检测铁凋亡相关蛋白和RAS/MAPK信号通路的表达。在裸小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细胞系具有显著较高的FMRP表达水平。生物信息学分析提示FMRP参与活性氧的调节,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,细胞GSH含量降低，MDA和ROS水平增加，Fe2+荧光强度,和线粒体膜电位，SLC7A11/GPX4蛋白表达和ERK磷酸化水平降低,MEK,MAPK,和RAS蛋白；FMRP过表达导致细胞发生相反的变化。在荷瘤裸鼠中，具有FMRP敲低的HCT116细胞显示出减弱的致瘤潜能,在异种移植物中降低的异种移植物生长速率和降低的SLC7A11表达。
结论：FMRP高表达抑制CRC细胞铁凋亡，通过激活RAS/MAPK信号通路促进CRC进展。
方法：我们使用RT-qPCR和Western印迹检测了CRC细胞系中FMRP的表达水平，并使用TCGA数据库分析了FMRP介导的CRC进展调控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个敲低载体(siFMRP-1、siFMRP-2和siFMRP-3),并使用CCK8法和平板克隆形成法检测其对HCT116细胞增殖的影响；使用MDA/ROS/GSH/Fe2试剂盒测定细胞铁凋亡水平的变化，使用JC-1荧光染色检测线粒体膜电位变化，免疫印迹法检测铁凋亡相关蛋白和RAS/MAPK信号通路的表达。在裸小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细胞系具有显著较高的FMRP表达水平。生物信息学分析提示FMRP参与活性氧的调节,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,细胞GSH含量降低，MDA和ROS水平增加，Fe2+荧光强度,和线粒体膜电位，SLC7A11/GPX4蛋白表达和ERK磷酸化水平降低,MEK,MAPK,和RAS蛋白；FMRP过表达导致细胞发生相反的变化。在荷瘤裸鼠中，具有FMRP敲低的HCT116细胞显示出减弱的致瘤潜能,在异种移植物中降低的异种移植物生长速率和降低的SLC7A11表达。
结论：FMRP高表达抑制CRC细胞铁凋亡，通过激活RAS/MAPK信号通路促进CRC进展。
