The N6-methyladenosine modification enhances ferroptosis resistance through inhibiting SLC7A11 mRNA deadenylation in hepatoblastoma

Abstract

Background: Solute carrier family 7 member 11 (SLC7A11) is overexpressed in multiple human tumours and functions as a transporter importing cystine for glutathione biosynthesis. It promotes tumour development in part by suppressing ferroptosis, a newly identified form of cell death that plays a pivotal role in the suppression of tumorigenesis. However, the role and underlying mechanisms of SLC7A11-mediated ferroptosis in hepatoblastoma (HB) remain largely unknown.

Methods: Reverse transcription quantitative real-time PCR (RT-qPCR) and western blotting were used to measure SLC7A11 levels. Cell proliferation, colony formation, lipid reactive oxygen species (ROS), MDA concentration, 4-HNE, GSH/GSSG ratio and cell death assays as well as subcutaneous xenograft experiments were used to elucidate the effects of SLC7A11 in HB cell proliferation and ferroptosis. Furthermore, MeRIP-qPCR, dual luciferase reporter, RNA pulldown, RNA immunoprecipitation (RIP) and RACE-PAT assays were performed to elucidate the underlying mechanism through which SLC7A11 was regulated by the m6A modification in HB.

Results: SLC7A11 expression was highly upregulated in HB. SLC7A11 upregulation promoted HB cell proliferation in vitro and in vivo, inhibiting HB cell ferroptosis. Mechanistically, SLC7A11 mRNA exhibited abnormal METTL3-mediated m6A modification, which enhanced its stability and expression. IGF2 mRNA-binding protein 1 (IGF2BP1) was identified as the m6A reader of SLC7A11, enhancing SLC7A11 mRNA stability and expression by inhibiting SLC7A11 mRNA deadenylation in an m6A-dependent manner. Moreover, IGF2BP1 was found to block BTG2/CCR4-NOT complex recruitment via competitively binding to PABPC1, thereby suppressing SLC7A11 mRNA deadenylation.

Conclusions: Our findings demonstrated that the METTL3-mediated SLC7A11 m6A modification enhances HB ferroptosis resistance. The METTL3/IGF2BP1/m6A modification promotes SLC7A11 mRNA stability and upregulates its expression by inhibiting the deadenylation process. Our study highlights a critical role of the m6A modification in SLC7A11-mediated ferroptosis, providing a potential strategy for HB therapy through blockade of the m6A-SLC7A11 axis.

Keywords: IGF2BP1; SLC7A11; ferroptosis; hepatoblastoma; m6A methylation; resistance.

FIGURE 1

Solute carrier family 7 member 11 (SLC7A11) promotes proliferation and mediates ferroptosis in HepG2 cells. (A) Relative expression of SLC7A11 in human HB and matched normal tissues was determined via RT‐qPCR (n = 35). HepG2 cells were transfected with siRNAs targeting SLC7A11, and silencing efficiency was verified via RT‐qPCR (B) and western blotting (C). HepG2 cells transfected with SLC7A11 siRNA#1 or SLC7A11 siRNA#2 were subjected to CCK8 assays (D) and colony formation assays (E) to evaluate the role of SLC7A11 in HB cell viability and proliferation. (F) HepG2 cells were treated with SLC7A11 siRNA#1 or SLC7A11 siRNA#2 for 48 h, and lipid ROS levels were then measured via BODIPY C11 staining coupled with flow cytometry. The relative GSH/GSSG ratio (G) and malondialdehyde (MDA) concentration (H) in HepG2 cells, which were transfected with SLC7A11 siRNA#1 or SLC7A11 siRNA#2 for 48 h, were detected using GSH, GSSG, and lipid peroxidation assay kits, respectively. (I) HepG2 cells were treated with SLC7A11 siRNA#1 or SLC7A11 siRNA#2 for 60 h, and cell death was then determined via propidium iodide (PI) staining coupled with flow cytometry. HepG2 cells transfected with SLC7A11 siRNA#1 or SLC7A11 siRNA#2 were treated with DMSO, ferrostatin‐1, erastin, erastin + ferrostatin‐1, erastin + ZVAD‐FMK, or erastin + necrosulfonamide, respectively, and lipid ROS levels (J) as well as cell death (K) were then determined via flow cytometry. HepG2 cells transfected with SLC7A11 siRNA#1 or SLC7A11 siRNA#2 were treated with DMSO, erastin, or erastin + GSH, respectively, and the levels of lipid ROS (L) as well as cell death (M) were then determined via flow cytometry. Erastin: 30 μM, Ferrostatin‐1: 1 μM, ZVAD‐FMK: 10 μM, Necrosulfonamide: 1 μM, GSH: 0.8 mM. Ctrl, control. All quantitative data are shown as the mean ± SD from three independent experiments. n.s., no significant difference, *p < .05, **p < .01, ***p < .001, ****p < .0001

FIGURE 2

METTL3 mediates the m6A modification of SLC7A11 mRNA, enhancing its stability and expression. (A) Pearson's correlation analysis indicated a positive correlation between METTL3 and SLC7A11 expression (n = 16) (r  =  0.7279, p  =  0.0014). The ΔΔCt values obtained from tumour‐normal tissue pairs (n = 16) were applied for Pearson's correlation analysis. (B) Relative expression of SLC7A11 in HuH6 and HepG2 cells upon METTL3 knockdown was determined via RT‐qPCR. (C–D) The relative m6A enrichment levels at the indicated site within the SLC7A11 mRNA 3′UTR was verified via meRIP‐qPCR analysis in HuH6 and HepG2 cells with or without METTL3 knockdown. (E) The decay rate of SLC7A11 mRNA in HuH6 and HepG2 cells with or without METTL3 depletion upon ActD (5 μg/ml) treatment was measured via RT‐qPCR at the indicated time points. (F) A schematic presentation of the pmir‐GlO luciferase reporters containing WT and Mut (GGAC to GGCC) SLC7A11 mRNA 3′UTR. (G) MeRIP‐qPCR was used to verify m6A levels of WT and Mut pmir‐GlO plasmids. (H) Luciferase activities of the WT and Mut pmir‐GlO plasmids were measured in HuH6 and HepG2 cells with or without METTL3 knockdown. All quantitative data are presented as the mean ± SD from three independent experiments. WT, wide‐type; Mut, mutation; n.s., no significant difference; ***p < .001

FIGURE 3

IGF2BP1 recognizes the m6A modification of SLC7A11 mRNA and enhances its stability and expression. (A) A schematic presentation of the SLC7A11 probes with methylated or unmethylated adenosine for screening the m6A reader of SLC7A11. (B) RNA pulldown followed by western blotting for endogenous IGF2BP protein screening in HuH6 cell lysates incubated with synthetic SLC7A11 probes (A probe, m6A probe and UTR‐NC probe). (C) Relative expression of IGF2BP1 in human HB and matched normal tissues was determined via RT‐qPCR (n = 16). (D) The Pearson's correlation coefficient was used to evaluate the correlation between IGF2BP1 and SLC7A11 expression in HB tissues (n  =  16) (r  =  0.7604, p  <  .001). (E) RIP‐qPCR results show the association of SLC7A11 3′UTR with IGF2BP1 in HuH6 and HepG2 cells. (F) RIP‐qPCR was used to detect the binding of IGF2BP1 and SLC7A11 3′UTR in HuH6 and HepG2 cells upon METTL3 knockdown. (G) Reduced SLC7A11 mRNA half‐life under IGF2BP1 silencing in HuH6 and HepG2 cells. Expression levels of SLC7A11 mRNA and protein upon IGF2BP deletion were determined via RT‐qPCR (H) and western blotting (I). (J) Relative luciferase activities of WT and Mut pmir‐GlO reporters were measured in HuH6 and HepG2 cells with or without IGF2BP knockdown. IP, immunoprecipitation; Ctrl, control; WT, wide type; Mut, mutation. All quantitative data are presented as the means ± SD of three independent experiments. n.s., no significant difference; **p  <  .01

FIGURE 4

IGF2BP1 competitively binds to PABPC1 to inhibit the deadenylation of SLC7A11 mRNA regulated by the BTG2/CCR4‐NOT complex. Poly(A) tail length of the endogenous SLC7A11 transcript under IGF2BP1 overexpression (A) or deletion (B) was determined via the RACE‐PAT assay in HuH6 and HepG2 cells. (C) Dual luciferase reporter assay showed the increased relative luciferase activities of WT pmir‐GlO reporter upon CAF1, CCR4A, or CNOT1 overexpression compared to Mut pmir‐GlO reporter in HuH6 and HepG2 cells. (D) Overexpressing IGF2BP1 enhanced the relative luciferase activities of the WT pmir‐GlO reporter under CAF1, CCR4A, or CNOT1 overexpression compared to Mut pmir‐GlO reporter in HuH6 and HepG2 cells. (E) Co‐immunoprecipitation of PABPC1 with BTG2 in HuH6 and HepG2 cells under IGF2BP1 knockdown. F. Relative expression of BTG2 in human HB and matched normal tissues was determined via RT‐qPCR (n = 16). (G) The mRNA levels of SLC7A11 under BTG2 gradient expression and IGF2BP1 deficiency were measured via RT‐qPCR in HuH6 and HepG2 cells. IP, immunoprecipitation; Ctrl, control; WT, wide type; Mut, mutation. All quantitative data are presented as the means ± SD of three independent experiments. *p < 0.05, **/##p  <  0.01

FIGURE 5

Knocking down METTL3 enhances the sensitivity of HB cells to ferroptosis. HuH6 and HepG2 cells transfected with METTL3 siRNA#1 were treated with erastin and lipid ROS levels (A) as well as cell death (B) were then measured via flow cytometry. HuH6 and HepG2 cells transfected with METTL3 siRNA#1 were treated with ferrostatin‐1, erastin, erastin + ferrostatin‐1, erastin + ZVAD‐FMK, or erastin + necrosulfonamide, respectively, and lipid ROS levels (C) as well as cell death (D) were then measured via flow cytometry. (E) Tumour images of the resected sh‐NC or sh‐METTL3‐1 tumours treated with or without IKE. (F) Tumour volumes were measured using an electronic calliper every 2 days and calculated using the formula volume (mm³) = Length (mm) × Width (mm²)/2. (G) Tumour weights of the resected sh‐NC or sh‐METTL3‐1 tumours treated with or without IKE. The proliferation of HuH6 and HepG2 cells stably transfected with sh‐METTL3 under SLC7A11 overexpression was assessed via CCK8 (H) and colony formation assays (I). The levels of lipid ROS (J) and cell death (K) in HuH6 and HepG2 cells stably transfected with sh‐METTL3 upon SLC7A11 overexpression were measured via flow cytometry. Erastin: HepG2 (30 μM) and HuH6 (20 μM), Ferrostatin‐1: 1 μM, ZVAD‐FMK: 10 μM, Necrosulfonamide: 1 μM, GSH: 0.8 mM. Ctrl, control; OV, overexpression vector. All quantitative data are presented as the means ± SD of three independent experiments. n.s., no significant difference, *p < .05, **p < .01, ***p < .001, ****p < .0001

FIGURE 6

A diagram illustrating the underlying molecular mechanism identified in this study. The diagram depicts the m6A‐dependent regulation of SLC7A11 mRNA that promotes the ferroptosis resistance of HB cells. Mechanistically, IGF2BP1 recognizes the METTL3‐mediated m6A modification of SLC7A11 mRNA and sustains its stability and expression through competitively binding to PABPC1. The interaction between IGF2BP1 and PABPC1 blocks recruitment of the BTG2/CCR4‐NOT complex to PABPC1 and inhibits the deadenylation of SLC7A11 mRNA