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

doi:10.12122/j.issn.1673-4254.2024.05.10

Abstract

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. Conclusion The high expression of FMRP inhibits ferroptosis in CRC cells and promotes progression of CRC by activating the RAS/MAPK signaling pathway.

Key words: fragile X mental retardation protein, ferroptosis, RAS/MAPK signaling pathway, colorectal cancer

Nan WANG, Bin SHI, Xiaolan MAN, Weichao WU, Jia CAO. High expression of fragile X mental retardation protein inhibits ferroptosis of colorectal tumor cells by activating the RAS/MAPK signaling pathway[J]. Journal of Southern Medical University, 2024, 44(5): 885-893.

Figures/Tables 8

Tab.1 Sequences of the small interfering RNAs

Gene	Sense (5'-3')	Antisense (5'-3')
hFMR1-1	GAGGAUGAUAAAGGGUGAGUUTT	AACUCACCCUUUAUCAUCCUCTT
hFMR1-2	CGAGAUUUCAUGAACAGUUUATT	UAAACUGUUCAUGAAAUCUCGTT
hFMR1-3	GCGUUUGGAGAGAUUACAAAUTT	AUUUGUAAUCUCUCCAAACGCTT

Tab.2 Antibody information

Antibody name	Company	Dilution rate
Anti-GAPDH	Cell Signaling technology	1∶1000
Anti-FMRP	Abcam	1∶1000
Anti-HO-1	Abmart	1∶1000
Anti-GPX4	Abmart	1∶1000
Anti-SLC7A11	Abmart	1∶1000
Anti-ERK	Cell signaling technology	1∶1000
Anti-p-ERK	Cell signaling technology	1∶1000
Anti-MAPK	Cell signaling technology	1∶500
Anti-p-MAPK	Cell signaling technology	1∶1000
Anti-RAS	Cell signaling technology	1∶500
Anti-p-RAS	Cell signaling technology	1∶500
Anti-MEK	Cell signaling technology	1∶1000
Anti-p-MRK	Cell signaling technology	1∶1000

Fig.1 Expression levels of FMRP in colorectal cancer (CRC) cell lines and tissues. A: RT-PCR for detecting the expression of FMRP in different CRC cell lines. B: Western blotting for detecting FMRP protein expression in different CRC cell lines. C: Expression of FMRP in colorectal cancer tissues and normal tissues. D: Relationship between FMRP expression and disease-free survival of CRC patients. *P<0.05 vs NCM460 group.

Fig.2 Bioinformatic analysis of biological functions and signaling pathways enriched in FMRP. A: Two groups of differential genes. B: Up-regulated and down-regulated differential genes. C: GO functional enrichment bands. D: GSEA-GO enrichment analysis. E: Circular enrichment map of differential genes in iron ion homeostasis. F: GSEA-KEGG analysis map.

Fig.3 Effect of FMRP knockdown or overexpression on HCT116 cell proliferation. A: Western blotting for verifying the efficiency of FMRP knockdown or overexpression. B: RT-PCR of FMRP mRNA expression in HCT116 cells. C: CCK8 assay of the proliferation of the transfected HCT116 cells. D: Plate clone formation assay of cell proliferation. *P<0.05, **P<0.01 vs Control or Lv-NC group.

Fig.4 Effect of FMRP knockdown or overexpression on ferroptosis of HCT116 cells. A: GSH contents in transfected HCT116 cells. B: MDA contents in the transfected cells. C: ROS level in the transfected cells. D: Fe2+ level in the transfected cells (scale bar=50 μm). E: Western blots of the ferroptosis markers in the transfected cells. F: Immunofluorescence detection of SLC7A11 in HCT116 cells with FMRP knockdown (scale bar=20 μm). G: Immunofluorescence detection of mitochondrial membrane potential in HCT116 cells with FMRP knockdown (scale bar=20 μm). *P<0.05, **P<0.01 vs Control or Lv-NC group.

Fig.5 Effect of FMRP knockdown on growth of transplanted tumor in nude mice. A: The size of subcutaneously transplanted tumor in nude mice. B: Changes in tumor weight in nude mice. C: Changes in tumor volume in nude mice. D: Immunohistochemistry for FMRP and SLC7A11 in the tumor tissue (scale bar=250 μm). *P<0.05, **P<0.01 vs Control.

Fig.6 Effect of FMRP knockdown or overexpression on RAS/MAPK signaling pathway in HCT116 cells detected by Western blotting. *P<0.05, **P<0.01 vs Control or Lv-NC group.

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