The m6A reader IGF2BP2 promotes the progression of myelodysplastic syndromes by inhibiting ferroptosis through stabilizing GPX4 mRNA, which can be reversed by CWI1-2
- 1Department of Clinical Laboratory， First Medical Center， Chinese PLA General Hospital， Beijing Chinese PLA Medical School， Beijing， China
- 2Department of Clinical Laboratory， First Medical Center， Chinese PLA General Hospital， Beijing
Abstract
Objective Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), as an N6-methyladenosine (m6A) reader protein, plays a role in various hematological malignancies. This study aimed to determine the effects of IGF2BP2 on oxidative stress and ferroptosis in myelodysplastic syndromes (MDS) and its underlying mechanism. Methods Bioinformatics analysis of Gene Expression Omnibus (GEO) datasets was performed to screen differentially expressed m⁶A-related genes between healthy control (HC) and MDS patients. Meanwhile, bone marrow samples from 15 MDS patients newly diagnosed in Department of Hematology, First Medical Center of Chinese PLA General Hospital from January 2023 to December 2023 were collected as the MDS group, and bone marrow samples from 12 healthy donors during the same period were collected as HC group. The identified target IGF2BP2 was further verified in clinical samples and the MDS cell lines SKM-1 and MUTZ-1. Dot blot assay was performed to determine m⁶A modification levels in the MDS group and in SKM-1 and MUTZ-1 cells. Small interfering RNA (siRNA) was used to establish an IGF2BP2-knockdown group (IGF2BP2-KD) and a negative control group (IGF2BP2-NC) in SKM-1 cells, and cell proliferation was detected using EdU fluorescence staining, CCK-8 assay, and cell cycle analysis. Western blotting and RT-qPCR were employed to detect the knockdown efficiency of IGF2BP2 and changes in the expression of glutathione peroxidase 4 (GPX4). RNA immunoprecipitation-quantitative PCR (RIP-qPCR) was performed to verify the interaction between IGF2BP2 and GPX4 mRNA, and GPX4 mRNA stability was assessed in IGF2BP2-NC and IGF2BP2-KD cells after actinomycin D treatment. To evaluate the intracellular oxidative stress levels associated with the GPX4 axis, flow cytometry was applied to measure reactive oxygen species (ROS), lipid peroxidation using BODIPY 581/591 C11, and myeloid differentiation markers CD14 and CD11b. Mitochondrial ultrastructural changes were observed with transmission electron microscopy (TEM), while glutathione (GSH) and malondialdehyde (MDA) contents and superoxide dismutase (SOD) activity were measured by visible spectrophotometry. A GPX4 overexpression rescue experiment was performed, with 4 groups set up: IGF2BP2-NC, NC+oeGPX4, IGF2BP2-KD, and KD+oeGPX4, to verify whether GPX4 mediates the effects of IGF2BP2 in MDS. In addition, SKM-1 cells were treated with the IGF2BP2-specific inhibitor CWI1-2 (CWI1-2 group) or vehicle control (CON group), and its effects on SKM-1 cell proliferation, differentiation, and oxidative stress were evaluated. Results Intersection analysis of the GSE19429 and GSE58831 datasets identified 8 differentially expressed factors (including 7 upregulated and 1 downregulated). Compared with the HC group, the m⁶A modification level improved, and the IGF2BP2 expression at mRNA and protein levels were significantly increased in the MDS group (P<0.01); same increases were also observed in SKM-1 and MUTZ-1 cells (P<0.05). EdU and CCK-8 assays showed that the IGF2BP2-KD group exhibited significantly inhibited SKM-1 cell proliferation (P<0.05) and G0/G1 cell cycle arrest (P=0.025 7) compared with the IGF2BP2-NC group. Western blotting and RT-qPCR demonstrated that IGF2BP2 knockdown significantly reduced GPX4 expression (P<0.01). RIP-qPCR indicated that the RIP-input ratio of GPX4 mRNA in the immunoprecipitates was significantly higher in the IGF2BP2 group than the IgG control group (P<0.000 1). Treatment with 5 μg/mL actinomycin D resulted in markedly decreased GPX4 mRNA level in the IGF2BP2-KD group than the IGF2BP2-NC group (P<0.000 1). The results of the flow cytometry analysis showed that compared with the IGF2BP2-NC group, the IGF2BP2-KD group showed significantly increased intracellular ROS levels and BODIPY 581/591 C11-detected lipid peroxidation(P<0.000 1, P<0.000 1), accompanied by decreased GSH content and total SOD activity (P<0.05) and increased MDA content (P=0.011 7). TEM revealed morphological alterations in the IGF2BP2-KD group, such as reduced or even absent mitochondrial cristae and increased mitochondrial membrane density. In the GPX4 overexpression rescue experiment, the KD+oeGPX4 group partially reversed the increases in ROS level and BODIPY 581/591 C11-detected lipid peroxidation, as well as the decreases in GSH content and SOD activity and the increment of MDA content in the IGF2BP2-KD group (P<0.05). After treatment with 1 μmol/L CWI1-2, the m⁶A-related function of IGF2BP2 was effectively inhibited, accompanied by a compensatory increase in IGF2BP2 protein level (P=0.001 1), whereas unchanged IGF2BP2 mRNA expression level (P=0.120 8), and decreased GPX4 protein and mRNA levels (P<0.05). EdU assay showed that CWI1-2 inhibited SKM-1 cell proliferation (P=0.003 8), while the expression levels of the myeloid differentiation markers CD14 and CD11b were significantly increased (P<0.01). In addition, the results of the flow cytometry analysis showed that CWI1-2 treatment significantly increased ROS levels and BODIPY 581/591 C11-detected lipid peroxidation (P<0.000 1, P<0.000 1), decreased GSH content and total SOD activity (P<0.001), and increased MDA content (P=0.001 4). Conclusion IGF2BP2 inhibits intracellular oxidative stress by regulating GPX4 expression and mRNA stability, thereby promoting MDS cell proliferation. CWI1-2, as an IGF2BP2 inhibitor, can promote ferroptosis through the IGF2BP2/GPX4 axis and possesses anti-MDS therapeutic potential.
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BibTeX
@article{yujie2026reader,
title = {{The m6A reader IGF2BP2 promotes the progression of myelodysplastic syndromes by inhibiting ferroptosis through stabilizing GPX4 mRNA, which can be reversed by CWI1-2}},
author = {Wei Yujie and Li Mianyang},
journal = {陆军军医大学学报},
year = {2026},
month = oct,
doi = {10.16016/j.2097-0927.202606096},
url = {https://doi.org/10.16016/j.2097-0927.202606096},
note = {CC BY},
}
RIS
TY - JOUR
TI - The m6A reader IGF2BP2 promotes the progression of myelodysplastic syndromes by inhibiting ferroptosis through stabilizing GPX4 mRNA, which can be reversed by CWI1-2
AU - Wei Yujie
AU - Li Mianyang
PY - 2026
DA - 2026/10/30
JO - 陆军军医大学学报
DO - 10.16016/j.2097-0927.202606096
UR - https://doi.org/10.16016/j.2097-0927.202606096
AB - Objective Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), as an N6-methyladenosine (m6A) reader protein, plays a role in various hematological malignancies. This study aimed to determine the effects of IGF2BP2 on oxidative stress and ferroptosis in myelodysplastic syndromes (MDS) and its underlying mechanism. Methods Bioinformatics analysis of Gene Expression Omnibus (GEO) datasets was performed to screen differentially expressed m⁶A-related genes between healthy control (HC) and MDS patients. Meanwhile, bone marrow samples from 15 MDS patients newly diagnosed in Department of Hematology, First Medical Center of Chinese PLA General Hospital from January 2023 to December 2023 were collected as the MDS group, and bone marrow samples from 12 healthy donors during the same period were collected as HC group. The identified target IGF2BP2 was further verified in clinical samples and the MDS cell lines SKM-1 and MUTZ-1. Dot blot assay was performed to determine m⁶A modification levels in the MDS group and in SKM-1 and MUTZ-1 cells. Small interfering RNA (siRNA) was used to establish an IGF2BP2-knockdown group (IGF2BP2-KD) and a negative control group (IGF2BP2-NC) in SKM-1 cells, and cell proliferation was detected using EdU fluorescence staining, CCK-8 assay, and cell cycle analysis. Western blotting and RT-qPCR were employed to detect the knockdown efficiency of IGF2BP2 and changes in the expression of glutathione peroxidase 4 (GPX4). RNA immunoprecipitation-quantitative PCR (RIP-qPCR) was performed to verify the interaction between IGF2BP2 and GPX4 mRNA, and GPX4 mRNA stability was assessed in IGF2BP2-NC and IGF2BP2-KD cells after actinomycin D treatment. To evaluate the intracellular oxidative stress levels associated with the GPX4 axis, flow cytometry was applied to measure reactive oxygen species (ROS), lipid peroxidation using BODIPY 581/591 C11, and myeloid differentiation markers CD14 and CD11b. Mitochondrial ultrastructural changes were observed with transmission electron microscopy (TEM), while glutathione (GSH) and malondialdehyde (MDA) contents and superoxide dismutase (SOD) activity were measured by visible spectrophotometry. A GPX4 overexpression rescue experiment was performed, with 4 groups set up: IGF2BP2-NC, NC+oeGPX4, IGF2BP2-KD, and KD+oeGPX4, to verify whether GPX4 mediates the effects of IGF2BP2 in MDS. In addition, SKM-1 cells were treated with the IGF2BP2-specific inhibitor CWI1-2 (CWI1-2 group) or vehicle control (CON group), and its effects on SKM-1 cell proliferation, differentiation, and oxidative stress were evaluated. Results Intersection analysis of the GSE19429 and GSE58831 datasets identified 8 differentially expressed factors (including 7 upregulated and 1 downregulated). Compared with the HC group, the m⁶A modification level improved, and the IGF2BP2 expression at mRNA and protein levels were significantly increased in the MDS group (P<0.01); same increases were also observed in SKM-1 and MUTZ-1 cells (P<0.05). EdU and CCK-8 assays showed that the IGF2BP2-KD group exhibited significantly inhibited SKM-1 cell proliferation (P<0.05) and G0/G1 cell cycle arrest (P=0.025 7) compared with the IGF2BP2-NC group. Western blotting and RT-qPCR demonstrated that IGF2BP2 knockdown significantly reduced GPX4 expression (P<0.01). RIP-qPCR indicated that the RIP-input ratio of GPX4 mRNA in the immunoprecipitates was significantly higher in the IGF2BP2 group than the IgG control group (P<0.000 1). Treatment with 5 μg/mL actinomycin D resulted in markedly decreased GPX4 mRNA level in the IGF2BP2-KD group than the IGF2BP2-NC group (P<0.000 1). The results of the flow cytometry analysis showed that compared with the IGF2BP2-NC group, the IGF2BP2-KD group showed significantly increased intracellular ROS levels and BODIPY 581/591 C11-detected lipid peroxidation(P<0.000 1, P<0.000 1), accompanied by decreased GSH content and total SOD activity (P<0.05) and increased MDA content (P=0.011 7). TEM revealed morphological alterations in the IGF2BP2-KD group, such as reduced or even absent mitochondrial cristae and increased mitochondrial membrane density. In the GPX4 overexpression rescue experiment, the KD+oeGPX4 group partially reversed the increases in ROS level and BODIPY 581/591 C11-detected lipid peroxidation, as well as the decreases in GSH content and SOD activity and the increment of MDA content in the IGF2BP2-KD group (P<0.05). After treatment with 1 μmol/L CWI1-2, the m⁶A-related function of IGF2BP2 was effectively inhibited, accompanied by a compensatory increase in IGF2BP2 protein level (P=0.001 1), whereas unchanged IGF2BP2 mRNA expression level (P=0.120 8), and decreased GPX4 protein and mRNA levels (P<0.05). EdU assay showed that CWI1-2 inhibited SKM-1 cell proliferation (P=0.003 8), while the expression levels of the myeloid differentiation markers CD14 and CD11b were significantly increased (P<0.01). In addition, the results of the flow cytometry analysis showed that CWI1-2 treatment significantly increased ROS levels and BODIPY 581/591 C11-detected lipid peroxidation (P<0.000 1, P<0.000 1), decreased GSH content and total SOD activity (P<0.001), and increased MDA content (P=0.001 4). Conclusion IGF2BP2 inhibits intracellular oxidative stress by regulating GPX4 expression and mRNA stability, thereby promoting MDS cell proliferation. CWI1-2, as an IGF2BP2 inhibitor, can promote ferroptosis through the IGF2BP2/GPX4 axis and possesses anti-MDS therapeutic potential.
SN - 2097-0927
ER -