EF24 targets METTL3 to reprogram m6A methylation and induce ferroptosis: an epitranscriptomic mechanism with therapeutical potential for glioma

EF24 targets METTL3 to reprogram m6A methylation and induce ferroptosis: an epitranscriptomic mechanism with therapeutical potential for glioma | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 July 2025 V1 Latest version Share on EF24 targets METTL3 to reprogram m6A methylation and induce ferroptosis: an epitranscriptomic mechanism with therapeutical potential for glioma Authors : YANG YANG , Zhou Jianxu , Liu Hao , Qu Nini , Li Ge , Yu Jingjie , and Piao Haozhe [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175154669.93436266/v1 Published Cell Communication and Signaling Version of record Peer review timeline 314 views 168 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Glioma, the most common primary malignant tumor of the central nervous system, still brings a dismal prognosis, even after stringent standard therapies. This study investigates the RNA epitranscriptomic regulatory mechanism of curcumin analog EF24 in inducing ferroptosis to counter glioma. Both in vitro and in vivo experiments demonstrated that EF24 induces significant ferroptosis and effectively suppresses the growth and metastasis of glioma. Mechanistically, EF24 specifically downregulates the m6A methyltransferase METTL3, a core component of the RNA methylation machinery. More specific, METTL3 recruits an m6A reader YTHDF1 to catalyze methylation at the 3’-UTR region of NRF2 mRNA, forming an m6A-YTHDF1-NRF2 translational enhancement complex that activates downstream antioxidant gene GPX4 expression. EF24 disrupts this complex through suppressing NRF2 mRNA stability and impairing its protein translation, ultimately depleting GPX4 and triggering ferroptosis cascades. For the first time, our study proposes an epitranscriptomic axis of METTL3/YTHDF1/NRF2/GPX4 as a regulator of ferroptosis in glioma, which may be targeted to design an effective and safe therapeutic strategy. 1. Introduction Glioma, the most prevalent primary intracranial tumor, originates from the overgrowth of neuroglial cells. 1 Stratified by the 2021 WHO Classification of Central Nervous System Tumors, grades 1-2 gliomas are designated as low-grade and grades 3-4 as high-grade. 2 Currently, the standard clinical management relies on surgical resection, followed by adjuvant radiotherapy and alkylating chemotherapy (e.g., temozolomide, TMZ), but cannot completely exclude the possibility of dismal survival outcomes. 3 This underscores the pressing need to explore molecular targets more specific and pharmacological agents more effective. Ferroptosis, a newly discovered form of programmed cell triggered by membrane damage, transforms the biochemical and morphological features of cells. Biochemically, ferroptosis induces intracellular iron overload, reactive oxygen species (ROS) accumulation, lipid peroxidation cascades, and functional inactivation of glutathione peroxidase (GPX) and the xCT system. 4 Morphologically, the nuclear architecture remaining largely intact under ferroptosis, but mitochondria display ultrastructural abnormalities, such as bilayer membrane density, cristae reduction or disappearance, and volume shrinkage. 5 Recent breakthroughs have demonstrated the therapeutic potential of ferroptosis induction in glioma management. For instance, betulinic acid suppresses glioma progression via inducing ferroptosis through inhibition on the PI3K/Akt pathway inhibition and stimulation on the NRF2/HO-1 axis. 6 Borax disrupts the HSPA5/NRF2/GPx4/GSH cytoprotective network to induce ferroptosis, thus selectively targeting to treat glioblastoma. 7 Therefore, the landscape of glioma treatment may be refreshed by a strategy that can target to effectively evoke ferroptosis. N6-methyladenosine (m6A) methylation, the most prevalent epitranscriptomic modification in eukaryotic RNA, is orchestrated by three classes of regulatory proteins: writers (methyltransferases), erasers (demethylases), and readers (RNA-binding proteins). 8 Among the writers, methyltransferase-like 3 (METTL3) serves as a powerful catalytic. Conversely, erasers, such as fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5), attenuate this modification. As multifunctional readers, the YT521-B homology (YTH) domain family (including YTHDF1/2/3 and YTHDC1/2) and insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1/2/3) can recognize m6A motifs and direct spatial-temporal regulation of target RNAs through compartmentalization or stability modulation. 9 Accumulating evidence demonstrates the implication of m6A dysregulation across a wide spectrum of malignancies. In a context-dependent manner, m6A regulators dynamically promote or suppress glioma development through fine-tuning critical processes, such as proliferation, migration, invasion, apoptosis, and cell cycle, thus offering an outcome highly m6A-related. 10 These findings may promise an m6A-oriented therapeutic strategy for glioma. Emerging evidence has unveiled an intricate crosstalk between m6A modification and ferroptosis. 11 As a post-transcriptional regulation, m6A epitranscriptomic reprogramming governs the susceptibility of malignancies to ferroptosis. 12 Notably, Y. Xu et al. demonstrated that METTL3 suppresses ferroptosis through m6A-mediated stabilization of SLC7A11 mRNA, thereby promoting lung adenocarcinoma progression. 13 Furthermore, C5aR1 potentiates METTL3-dependent GPX4 expression via ERK1/2 activation to inhibit ferroptosis, and drive the progression of glioblastoma multiforme (GBM). 14 Intriguingly, upon the EGFR signaling ALKBH5 demethylase is maintained in the nuclei to enhance global epitranscriptomic reprogramming, thus conferring glioblastoma with resistance to ferroptosis. 15 Collectively, these findings highlight the central role of m6A-driven ferroptosis in oncogenesis, which may be based on to design targeted therapies. Compared to its parent compound, EF24 (3,5-Bis(2-fluorobenzylidene)-4-piperidone), exhibits a superior anticancer efficacy, as a synthetic curcuminoid analogue with higher bioavailability, metabolic stability, and cellular uptake. 16 Preclinical studies have delved into its antitumor mechanisms. Yin et al. 17 demonstrated that EF24 suppresses cholangiocarcinoma progression through silencing the NF-κB signaling. Duan et al. 18 revealed its ability to curb the proliferation and invasion of triple-negative breast cancer cells via targeting the lncRNA HCG11/Sp1 axis. Notably, EF24 shows a ferroptosis-inducing potential, as evidenced by HMOX-1-mediated ferroptotic cell death in osteosarcoma models. 19 A 2021 Biomolecules study highlighted the anti-glioma activity of EF24 through phenotypic modulation. 20 However, the role of EF24 in modulating ferroptosis in glioma remains unexplored. In this study, we systematically investigated the impact of EF24 on m6A epitranscriptomic reprogramming and ferroptosis in glioblastoma multiforme (GBM) cells. Through mechanistic exploration, we verified the mediatory role of the METTL3/YTHDF1/NRF2/GPX4 regulatory axis in the antitumor effects of EF24. These findings provide the epitranscriptomic mechanisms in glioma pathobiology, and the therapeutic potential of EF24 against GBM. 2 Materials and Methods 2.1 Cell culture Human glioma cell lines LN229(RRID:CVCL-0393) and A172(RRID:CVCL-0131) were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA), cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin antibiotic cocktail (10,000 U/mL penicillin, 10 mg/mL streptomycin). Cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂. 2.2 Antibodies and reagents EF24, Actinomycin D (MCE), METTL3(Cat# METTL3, RRID:AB_3697867), YTHDF1(Cat# 80876-2-RR, RRID:AB_3670491), GPX4(Cat# CL488-82949, RRID:AB_3673149), Ki67(Cat# Ki67-1010, RRID:AB_2941673), 4HNE (Cat# 24325, RRID:AB_2716829), NRF2(Cat# NBP3-26113, RRID:AB_3638302), and GAPDH were used in this study. Secondary antibodies were obtained from HUABIO (Hangzhou, China). 6-Carboxy-2’,7’-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (DCFDA) was purchased from Invitrogen. Antibodies against GAPDH and additional secondary antibodies were acquired from Hua-an Biotechnology. 2.3 Cell counting kit-8 assay Cells were seeded in 96-well plates at a density of 3,000 cells per well and treated with EF24 at gradient concentrations (0.375, 0.75, 1.5, 3, 6, 9 μM) for 24, 48, or 72 hours. Afterward, 10 μL CCK-8 reagent was added to each well, followed by an incubation at 37°C for 2 hours. Absorbance was subsequently measured at 450 nm using a microplate reader. 2.4 Colony formation assay Cells were seeded in 6-well plates at a density of 1,500 cells per well and exposed to EF24 (1.5 μM, 3 μM) or vehicle control (0.05% DMSO). Following a 14-day incubation, cells were fixed with 4% paraformaldehyde (15 min, room temperature) and stained with 0.5% crystal violet solution (20 min). Colonies were gently rinsed with distilled water to remove excess stains, and then air-dried overnight. Quantitative analysis was performed by capturing digital images and enumerating colonies (>50 cells/colony) using ImageJ software (version 1.53) with threshold-based automatic particle analysis. 2.5 Immunofluorescence assay Glioma cells were plated on sterile glass coverslips in 12-well plates and allowed to adhere for 12 hours. The cells were subsequently treated with experimental compounds under specified conditions. After treatment, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature, followed by permeabilization with 0.2% Triton X-100 in PBS for 15 min. Nonspecific binding was blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Primary antibodies targeting 4-HNE (1:200 dilution; Abcam, Cat# ab48506) and Ki67 (1:250 dilution; Cell Signaling Technology, Cat# 9129) were applied overnight at 4°C. After three times of washing with PBS, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:500; Invitrogen, Cat# A-11034) for 1 h at room temperature in the dark. Nuclei were counterstained with 1 μg/mL DAPI (Beyotime Biotechnology, Cat# C1002) for 10 min. Coverslips were mounted using ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and imaged using a confocal fluorescence microscope (Zeiss LSM 880) with appropriate excitation/emission filters. 2.6 Quantitative real time PCR (qRT-PCR) Cellular RNA was collected using TRIzol Reagent (ABclonal, Wuhan, China), and reversely transcribed into cDNA using the ABScript Neo RT Master Mix for qPCR with gDNA Remover (ABclonal). For genes of interests, thermal reactions were performed using the BrightCycle Universal SYBR Green qPCR Mix with UDG at 37°C for 2 min and 95°C for 3 min, accompanied by 40 cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension at 95°C for 5 s and 60°C for 30 s on the CFX96 Touch™ Real- Time PCR system (Bio- Rad, Hercules, CA, USA). Relative expression levels were measured by the 2−ΔΔ CT method and normalized to that of GAPDH. The primer sequences used were as follows: METTL3: forward, 5′-CATTGCCCACTGATGCTGTG-3′; reverse, 5′-AGGCTTTCTACCCCATCTTGA-3′. YTHDF1: forward, 5′-CAAGCACACAACCTCCATCTTCG-3′; reverse, 5′-GTAAGAAACTGGTTCGCCCTCAT-3′. NRF2: forward, 5′-TTCCCGGTCACATCGAGAG-3′; reverse, 5′-TCCTGTTGCATACCGTCTAAATC-3′. GPX4: forward, 5′-CGCTGTGGAAGTGGATGAAG-3′; reverse, 5′-TTGTCGATGAGGAACTGTGG-3′. GAPDH: forward, 5′-ACATCGCTCAGACACCATG-3′; reverse, 5′-TGTAGTTGAGGTCAATGAAGGG-3′. The siRNA sequences used were as follows: siMETTL3-1: forward, 5′-CCUGCAAGUAUGUUCACUA-3′; reverse, 5′-UAGUGAACAUACUUGCAGG-3′. siMETTL3-2: forward, 5′-GCUCAACAUACCCGUACUA-3′; reverse, 5′-UAGUACGGGUAUGUUGAGC-3′. siYTHDF1-1: forward, 5′-GGCGUGUGUUCAUCAUCAUCAA-3′; reverse, 5′-UUGAUGAUGAACACACGCC-3′. siYTHDF1-2: forward, 5′-CCUGCUCUUCAGCGUCAAU-3′; reverse, 5′-AUUGACGCUGAAGAGCAGG-3′. The METTL3 and YTHDF1 overexpression plasmids were purchased from MiaoLingBio (China). The YTHDF1 plasmid (pLV3-CMV-YTHDF1(human)-3×FLAG-CopGFP-Puro) was constructed in the pLV3 vector, while the METTL3 plasmid (pCMV-METTL3(human)-3×FLAG-Puro) in the pCMV vector. 2.7 Immunohistochemistry Paraffin-embedded tissue sections were dewaxed using xylene and a gradient of ethanol solutions. After processing, the sections were blocked with goat serum and incubated with primary antibodies at 4°C overnight. Immunoperoxidase staining was performed using a DAB detection kit, and hematoxylin was applied for nuclear counterstaining. Finally, the sections were dehydrated through a graded ethanol series and sealed with neutral resin. 2.8 GSH and MDA assays Intracellular levels of glutathione (GSH) and malondialdehyde (MDA) were measured using the Glutathione (GSH) Assay Kit (Sigma, MAK364) and the Lipid Peroxidation (MDA) Assay Kit (Sigma, MAK085), respectively. All experimental procedures were performed according to the manufacturer’s instructions. 2.9 Reactive oxygen species (ROS) assays LN229 and A172 cells were seeded in 6-well plates at a density of 1×10⁶ cells per well and cultured until attachment. Fresh Hank’s Balanced Salt Solution (HBSS) containing 10 μM H2DCFH-DA probe (Invitrogen, Carlsbad, CA, USA) was then added to each well, followed by an incubation at 37°C for 30 min. Subsequently, the cells were resuspended in 400 μL HBSS and analyzed by flow cytometry. Data acquisition and analysis were performed using FlowJo V10 software. 2.10 Methylated RNA immunoprecipitation and qRT- PCR (MeRIP- qPCR) MeRIP- qPCR was performed with m6A RNA Methylation Quantification Kit (Epibiotek). Total RNA was fragmented and prepared for magnetic beads, which were then immunoprecipitated, eluted and purified. Purified RNA was finally analyzed by qRT-PCR. 2.11 RNA immunoprecipitation (RIP) assay The RIP assay was performed using EpiTM RNA immunoprecipitation (qRT-PCR) kit (Epibiotek) according to the manufacturer’s instructions. Briefly, the indicated cell lysates were collected and incubated with Protein A/G magnetic beads coated with control IgG antibody, anti-YTHDF1 antibody(abcam, ab220162) with rotation at 4℃ overnight. The next day, co-precipitated RNA was extracted by TRIzol Reagent (ABclonal, Wuhan, China) and Phenol-chloroform method. The relative expression of NRF2 was detected by RT-qPCR. IP enrichment was normalized to the input yielded from the same number of cells. 2.12 mRNA stability assay Transfected cells were seeded in 6-well plates and cultured. At a 70% confluence, the cells were treated with actinomycin D (15 μg/mL for LN229 cells and 10 μg/mL for A172 cells) for 0, 1.5, 2.5, and 3.5 h, followed by mRNA quantification using qRT-PCR. 2.13 Western blotting analysis RIPA buffer supplemented with protease inhibitors was used to extract total protein; protein quantitation was conducted using the BCA protein assay kit (Biosharp, China). Samples were electrophoresed on a 10% SDS polyacrylamide gel (SDS-PAGE), then transferred onto an NC membrane. The membrane was then blocked with 5% non-fat milk for 1 h, followed by washing, incubation with primary and secondary antibodies, and finally detected with enhanced chemiluminescence. A Nuclear Protein Extraction Kit (Solarbio, China) was used for nuclear and cytosol protein (NRF2,METTL3) extraction according to the manufacturer’s instructions. 2.14 Hematoxylin-eosin (H&E) staining Tumor specimens were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned (5 μm). After deparaffinization in xylene and graded ethanol hydration, sections were stained with hematoxylin, differentiated in 1% HCl-ethanol, counterstained with eosin, dehydrated, cleared in xylene, and mounted with neutral balsam. Images were acquired using a light microscope. 2.15 Animal experiments Stable cell lines with METTL3 gene overexpression in LN229-Luc were established to investigate the function of METTL3 in glioma in vivo. Female, BALB/c, nude, 4–5-week-old mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in individually ventilated cages (IVC) under specific-pathogen-free (SPF) conditions with a 12 h light/dark cycle at 23 ± 1 °C. All animals had free access to standard SPF mouse chow and water. The animal studies were approved by the Animal Ethics Committee of Dalian Medical University (AEE23105). The mouse skulls were exposed after anesthesia (pentobarbital sodium). A total of 5×10 5 LN229-Luc cells diluted in 5 μL PBS were injected into the right frontal lobe of each mouse: 0.5 mm anteriorly, 2.0 mm laterally, and 3.0 mm ventral coordinates relative to bregma. After injection, the needle was held in position for 5 min and then slowly withdrawn. The mice were kept in an environment with a light rhythm after wound saturation. The In Vivo Imaging System (IVIS) (Spectrum, PerkinElmer) was used to measure the LN229 tumor grafts in mouse brains. 2.16 Statistical analyses All cell experiments were conducted in triplicate. Continuous variables showing normal distribution (Shapiro-Wilk test, p>0.05) were expressed as mean ± SD. Between-group comparisons employed two-tailed Student’s t-test with Welch’s correction for unequal variances. Multiple comparisons were analyzed by one-way ANOVA followed by Tukey’s post hoc test for homogeneous variances or Games-Howell test for heterogeneous variances (Brown-Forsythe test p<0.05). All analyses were conducted in Prism 9 (GraphPad), with exact p-values reported (***p<0.001, **p<0.01, *p<0.05). 3. Results 3.1 EF24 inhibits glioma cell proliferation The viability of LN229 and A172 cells decreased significantly as EF24 concentration and treatment duration increased. After 24 h of EF24 treatment, the half-maximal inhibitory concentration (IC50) was determined as 4.494 μM for LN229 cells and 5.114 μM for A172 cells (Figure 1A). To assess the inhibitory effects of EF24 on glioma cell proliferation and colony formation, LN229 and A172 cells were treated with 1.5 μM and 3 μM EF24, respectively. The colony formation assay showed that in a concentration-dependent manner, EF24 reduced the number and size of colonies significantly, compared to the control group, indicating that EF24 can suppress glioma cell proliferation (Figure 1B). Following treatment with EF24 (0 μM, 1.5 μM, 3 μM)A, immunofluorescence assays were performed to evaluate the expression level of Ki67, a nuclear antigen indicating cell proliferation. The results revealed a marked reduction in Ki67 expression in both cell lines after EF24 treatment, further supporting its anti-proliferative effects (Figure 1C). Figure 1. EF24 Inhibits Glioma Cell Proliferation. (A) CCK-8 assay showing the inhibitory effect of EF24 on glioma cell viability and the IC50 values for LN229 and A172 cells. (B) Plate colony formation assay assessing the effect of EF24 on glioma cell proliferation. (C) Immunofluorescence analysis of the effect of EF24 on glioma cell proliferation (magnification ×200, scale bar: 50 μm). Note: *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group. 3.2 EF24 induces ferroptosis in glioma cells The immunofluorescence analysis revealed a significant increase in intracellular 4-hydroxynonenal (4-HNE) levels following EF24 treatment (0 μM, 1.5 μM, 3 μM) (Figure 2A). Glutathione (GSH) assays demonstrated that EF24 (0 μM, 1.5 μM, 3 μM) markedly reduced GSH levels in both A172 and LN229 cells (Figure 2B). Similarly, EF24 elevated malondialdehyde (MDA) levels significantly in both cell lines, compared to the control group (Figure 2C). To assess ROS accumulation, glioma cells were stained with the DCFH-DA fluorescent probe and analyzed by flow cytometry. EF24 increased the intensity of ROS fluorescence prominently, suggesting that EF24 promotes ROS accumulation and induces ferroptosis (Figure 2D). To further validate the successful induction of ferroptosis, mitochondrial morphology was examined using transmission electron microscopy (TEM). After a 24-h treatment with EF24 (3 μM), LN229 cells exhibited marked mitochondrial alterations, including reduced mitochondrial volume, thickened or ruptured mitochondrial membranes, and loss of cristae structure, compared to untreated controls (Figure 2E). Collectively, EF24 triggers ferroptosis in glioma cells. Figure 2. EF24 induces ferroptosis in glioma cells. (A) Immunofluorescence of intracellular 4-HNE following EF24 treatment (magnification ×200, scale bar: 200 μm).(B) Intracellular MDA levels following EF24 treatment. (C) Intracellular GSH levels following EF24 treatment. (D) DCFH-DA fluorescent probe analysis of ROS fluorescence intensity in cells following EF24 treatment. (E) Transmission electron microscopy (TEM) of mitochondrial morphological changes following EF24 treatment (magnification ×7000, scale bar: 2.0 μm; magnification ×20000, scale bar: 500 nm). Note: *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group. 3.3 METTL3 expression elevates in glioma tissues and cells METTL3 expression elevated significantly in human glioma tissues compared to normal brain tissues, with a level increasing grade. Furthermore, METTL3 was consistently overexpressed across multiple human glioma cell lines (Figure 3-1A–C). Figure 3. METTL3 Expression Elevates in Glioma Tissues and Cells. (A) METTL3 expression levels in normal brain tissues and glioma tissues. (B) METTL3 expression levels in different human glioma cell lines. (C) METTL3 expression levels in tissues and cell lines, respectively. Note: *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group. 3.4 METTL3 inhibits ferroptosis in glioma cells NRF2, a key antioxidant transcription factor, regulates ferroptosis by targeting a cascade of downstream molecules. Notably, NRF2 has been shown to inhibit GPX4 to trigger ferroptosis. To investigate whether METTL3 modulates the protein expression of NRF2 and GPX4, we transfected siMETTL3 and si-Control into LN229 and A172 cell lines to establish METTL3-knockdown and control cell models, respectively. Western blotting analysis confirmed a successful transfection (Figure 4A), and protein levels of NRF2 and GPX4 significantly reduced in METTL3-depleted cells, indicating that METTL3 post-transcriptionally regulates both genes (Figure 4A). Correlation analysis via Spearman’s rank correlation coefficient (GEPIA, http://gepia.cancer-pku.cn/) further revealed a positive association between METTL3 expression and the mRNA levels of NRF2 and GPX4 in glioblastoma (GBM) tissue specimens (Figure 4B). Collectively, these findings support NRF2 and GPX4 as downstream effectors of METTL3 in GBM cells. MDA assays revealed a statistically significant increase in intracellular MDA levels upon METTL3 knockdown (Figure 4C). Conversely, GSH levels in A172 and LN229 cells were markedly reduced following METTL3 depletion (Figure 4D). To further validate the inhibitory role of METTL3 in ferroptosis, TEM was employed to examine mitochondrial morphology in LN229 cells treated with siMETTL3 for 24 h. Compared to controls, METTL3 knockdown cells exhibited pronounced shrinkage of mitochondria, thickening or rupture of membranes, and loss of cristae (Figure 4E). In summary, METTL3 acts on its downstream NRF2 and GPX4 to modulate redox homeostasis and mitochondrial integrity, and METTL3 depletion can induce ferroptosis in glioma cells. Figure 4. METTL3 inhibits ferroptosis in glioma cells. (A) Western blotting analysis of NRF2 and GPX4 in LN229 and A172 cells in the negative control group and the METTL3 knockdown group. (B) Correlation analysis of METTL3 expression with NRF2 and GPX4 mRNA levels in GBM tissue specimens. (C) Detection of intracellular malondialdehyde (MDA) levels after METTL3 knockdown. (D) Intracellular glutathione (GSH) levels following METTL3 knockdown. (E) TEM images showing mitochondrial morphological changes in METTL3-knockdown cells (magnification: ×7000, scale bar = 2.0 μm; magnification: ×20000, scale bar = 500 nm).Note: * P < 0.05, ** P < 0.01, *** P < 0.001 versus the control group. 3.5 METTL3 undergoes m6A modification to stabilize NRF2 expression Forty-three m6A binding sites were found in the sequence of NRF2 based on SRAMP (Welcome to SRAMP, an online m6A site predictor) prediction, suggesting that NRF2 does undergo m6A modification (Figure 5A). meRIP-qPCR experiments showed that NRF2 was significantly enriched in the m6A antibody (Figure 5B). Actinomycin experiments revealed that knockdown of METTL3 reduced NRF2 stability (Figure 5C). Similarly knockdown of METTL3 decreased NRF2 mRNA expression (Figure 5D). Taken together, METTL3 is indeed involved in the m6A modification of NRF2. Figure 5 METTL3 undergoes m6A modification to stabilize NRF2 expression. (A) SRAMP website predicting the m6A site of NRF2. (B) MeRIP assay verifying d that NRF2 undergoes m6A modification. (C) Actinomycin assay detecting the effect of METTL3 on the stability of NRF2. (D) qRT-PCR detecting the effect of knocking down METTL3 on NRF2 expression. Note: Compared with the control group, *P<0.05, **P<0.01, ***P<0.001. 3.6 METTL3 recruits YTHDF1 to regulate NRF2 mRNA stability Previous studies have shown that METTL3 can recruit YTHDF1 to enhance the stability of its target transcripts. Here, actinomycin assay revealed that knockdown of YTHDF1 reduced NRF2 stability (Figure 6A). Similarly, knockdown of YTHDF1 decreased NRF2 mRNA expression (Figure 6B). It can be hypothesized that YTHDF1 is involved in the m6A modification of NRF2. Correlation analysis by Spearman’s rank correlation coefficient (GEPIA, http://gepia.cancer-pku.cn/) showed that YTHDF1 was positively correlated with NRF2 expression in GBM tissue specimens (Figure 6C). Western blotting analysis showed that YTHDF1 silencing inhibited NRF2 protein level (Figure 6D). In addition, RIP-qPCR analysis showed that YTHDF1 directly bound to NRF2 mRNA, and METTL3 knockdown significantly reduced this binding efficiency, suggesting that METTL3 blocks the direct interaction between YTHDF1 and NRF2 mRNA (Figure 6E). Taken together, methylated NRF2 mRNA is recognized by YTHDF1, and METTL3 employs YTHDF1 to enhance NRF2 mRNA stability. Figure 6 METTL3 recruits YTHDF1 to regulate NRF2 mRNA stability. (A) Actinomycin assay showing the effect of YTHDF1 on NRF2 stability. (B) qRT-PCR showing the effect of knockdown of YTHDF1 on NRF2 expression. (C) Correlation analysis of YTHDF1 and NRF2 expression in GBM tissue specimens. (D) Western blotting showing the effect of knockdown of YTHDF1 on NRF2 expression. (E) RIP-qPCR revealing the binding of YTHDF1 with NRF2 in stable METTL3 knockdown and negative control LN229 and A172 cells. Note: * P < 0.05, ** P < 0.01, *** P < 0.001 compared to control. 3.7 EF24 induces ferroptosis in glioma cells via the METTL3/YTHDF1/NRF2/GPX4 axis Western blotting and qRT-PCR results revealed that the protein and mRNA levels of METTL3, YTHDF1, NRF2 and GPX4 were significantly downregulated in LN229, A172 cells after EF24 treatment (Figure 7A-B). To further confirm that EF24 directly acts on METTL3, we successfully transfected METTL3 overexpression (OE) and vector plasmids into LN229 and A172 cell lines, respectively. The protein replication assay found that vector+EF24 reduced protein expression and mRNA levels of YTHDF1, NRF2, and GPX4, but OE METTL3+EF24 reverted these changes, confirming that EF24 could act directly on METTL3 (Figure 7C-D). To verify that EF24 could regulate the m6A modification of NRF2 via METTL3, we performed MeRIP and actinomycin experiments, which demonstrated that EF24 treatment significantly decreased the m6A levels, as well as the mRNA level of NRF2 in LN229 and A172 cells, indicating that EF24 could regulate the m6A modification of NRF2 through METTL3 (Figure 7E-F). NRF2 undergoes oxidative stress and METTL3 undergoes methylation in the nucleus. The nucleoplasmic separation assay showed that EF24 decreased the expression of both NRF2 and METTL3 in the cytoplasm and nucleus, suggesting that EF24 can demonstrate actions in the nucleus (Figure 7G). Figure 7 EF24 induces ferroptosis in glioma cells via the METTL3/YTHDF1/NRF2/GPX4 axis. (A) METTL3, NRF2 and GPX4 protein levels after EF24 treatment. (B) METTL3, NRF2 and GPX4 mRNA levels after EF24 treatment. (C) Overexpression of METTL3 in the protein replication assay. (D) Overexpression of METTL3 in the mRNA replication assay. (E) MeRIP assay verifying that EF24 can interfere with m6A modification. (F) Actinomycin assay detecting the effect of EF24 on NRF2 stability. (G) NRF2 and METTL3 expression in cytoplasm and nucleus after EF24 treatment. Note: Compared with the control group, *P<0.05, **P<0.01, and ***P<0.001. 3.8 The ferroptosis-inducing mechanism of EF24 is validated in the mouse model To further validate the ferroptosis-inducing mechanism of EF24, we first constructed the LN229-luc cell line with OE METTL3 and vector. Western blotting was performed to validate the efficiency of OE METTL3 transfection (Figure 8A). LN229-luc cells (vector, OE METTL3) were injected into the lateral ventricles of nude mice by a brain stereotaxic apparatus, and three groups of in situ glioma nude mice were constructed, including vector group, vector+EF24 group, and OE METTL3+EF24 group, with 10 nude in each group (5 for tumor size analysis and 5 for survival analysis) (Figure 8B). We observed, photographed and recorded the sizes of in situ glioma by an in vivo imaging system (IVIS) on the 10th, 17th, and 24th days after the successful seeding of the tumor, respectively. The data showed that the tumor size increased slowly in the vector+EF24 group, and EF24 significantly inhibited the tumor growth, as compared with those in the vector and OE METTL3+EF24 groups (Figure 8C.). The survival period was significantly prolonged in the vector+EF24 group, and the weight loss was significantly attenuated, compared with those in the vector and OE METTL3+EF24 groups (Figure 8D-E). According to the 3R principle of animal experiments, we carried out the extraction treatment on the 24th day after the successful tumor seeding. Tumor tissues were embedded, sectioned, and stained after extraction. H&E staining showed that the maximum diameter and cross-sectional area of tumors in the vector+EF24 group were significantly smaller than those in the vector group and the OE METTL3+EF24 group, suggesting that EF24 could prevent the growth of tumors in mice (Figure 8F). Finally, we evaluated the possible detrimental effects of EF24 on vital organs. The heart, liver, spleen, lung and kidney tissues were taken from nude mice after euthanasia. H&E staining showed no significant damage to these organs in nude mice (Figure 8G). We further performed immunohistochemical staining and Western blotting experiments on tumor tissue sections from tumor-bearing nude mice. Immunohistochemical staining results showed that the vector+EF24 group METTL3, YTHDF1, NRF2, GPX4 had lower mean optical density values than the vector group, while these values were increased in the OE METTL3+EF24 group (Fig. 8H). Western blotting results showed that, compared with those in the vector group, the METTL3, YTHDF1, NRF2, GPX4 levels were significantly downregulated in the vector+EF24 group, and the YTHDF1, NRF2, and GPX4 levels were reversed in the OE METTL3+EF24 group (Figure 8I). GSH and MDA levels were also detected in tumor tissue samples, and the results showed that compared with those in the vector group, GSH level and MDA accumulation fell in the vector+EF24 group, but then rose in the METTL3+EF24 group (Figure 8J). TEM results showed typical ferroptosis-related morphological characteristics that the mitochondria in the vector+EF24 group (Figure 8K). Consistent with in vitro results, our in vivo validation experiments suggest that EF24 can induce ferroptosis through the METTL3/YTHDF1/NRF2/GPX4 axis to prevent glioma growth. Figure 8 The ferroptosis-inducing mechanism of EF24 is validated in the mouse model. (A) Western blotting showing OE METTL3 transfection efficiency. (B) Construction of in situ brain glioma model in nude mice. (C) The IVIS to compare the fluorescence intensity of tumor in nude mouse brain. (D) Survival curves of nude mice in the three groups. (E) Body weight curves of nude mice in the three groups. (F) Comparison of tumor diameter and cross-sectional area in tumor-bearing mice by H&E staining (magnification ×20, scale bar = 500 μm, magnification ×200, scale bar = 50 μm). (G) H&E staining observing the damage of EF24 to various organs in nude mice (scale bar = 100 μm). (H) Immunohistochemistry detecting the expression levels of METTL3, YTHDF1, NRF2 and GPX4 in tumor tissue sections of nude mice in each group, and the average optical density values were calculated and counted by using Image J software (magnification × 200, scale bar = 50 μm). (I) Western blotting detecting the expression levels of METTL3, YTHDF1, NRF2 and GPX4 in tumor tissue sections of nude mice in each group. (J) GSH and MDA levels in each group. (K) TEM displaying the mitochondrial morphology in tumor tissues of nude mice. Yellow markers indicate normal mitochondria, red markers indicate iron-dead mitochondria (magnification × 30,000, scale bar = 0.5 μm). Note: *P<0.05, **P<0.01, ***P<0.001. 4. Discussion EF24, a synthetic monocarbonyl curcumin analog, demonstrates significant chemopreventive and chemotherapeutic potentials against various tumors, 21 including breast cancer, lung cancer, liver cancer, gastric cancer, and GBM. 22 In recent years, ferroptosis, a newly discovered form of cell death, has also slipped into the spotlight of tumor-related research. 23 Unlike traditional apoptosis, autophagy and necrosis, ferroptosis is influenced by GPX4 inhibition and closely associated with the accumulation of iron-dependent lipid peroxides. 24 Increasing evidence has revealed the unique characteristics of iron metabolism in glioma cells. Therefore, molecules involved in ferroptosis may be targeted to revolutionize glioma treatment. 25 Here, we for the first time reported the role and mechanism of EF24 in promoting ferroptosis in glioma cells, thus offering a candidate for future treatment designs. We examined the expression level of the proliferation-associated nuclear antigen Ki67 in glioma cells treated with EF24 using clonogenic and immunofluorescence assays. The results showed that EF24 significantly inhibited the expression of Ki67, suggesting EF24 as a therapeutic agent for glioma. Ferroptosis elicits series characteristic cellular processes, including a decrease in intracellular GSH levels, an accumulation of ROS, and an increase in lipid peroxidation products, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). These changes ultimately lead to mitochondrial shrinkage, membrane hyperdensity, and cristae reduction. 26 Our results demonstrated that EF24 treatment induced corresponding changes in GSH, MDA, ROS, and mitochondrial morphology. This study is the first to reveal that EF24 induces glioma cell death through a ferroptosis-dependent mechanism. METTL3 catalyzes the methylation of RNA by transferring a methyl group from S-adenosylmethionine (SAM) to form m6A. Due to this m6A-enabling effect, METTL3 has been demonstrated closely associated with the occurrence and progression of various malignancies. For instance, METTL3 knockout halts the proliferation, survival, and invasiveness of human lung cancer HeLa cells. 27 Additionally, METTL3 is significantly upregulated in bladder cancer, and its depletion markedly decreases bladder cancer cell proliferation, invasion, in vitro survival, and in vivo tumorigenicity. 28 In this study, we found that METTL3 was highly expressed across different human glioma cell lines. Moreover, METTL3 expression levels correlated positively with glioma grade, consistent with previous studies. 29 In the METTL3 knockdown model, we observed dysmetabolism of intracellular GSH, accumulation of lipid peroxidation products, and ultrastructural changes in mitochondria, suggesting that METTL3 inhibits ferroptosis in glioma cells. This finding provides a novel perspective on the role of METTL3-mediated m6A modification in the regulation of ferroptosis. NRF2, encoded by NFE2L2 gene, is a key transcription factor regulating oxidative stress. It binds to the antioxidant response element (ARE) in the promoter regions of downstream target genes, thereafter activating the expression of antioxidant and ferroptosis-related genes, including SLC7A11, GPX4, and FTH1. Nrf2 maintains redox homeostasis to protect normal cells; on the other hand, its excessive activation enhances the resistance of tumor cells to ferroptosis, thus promoting tumor progression. 30 Our study found that METTL3 knockdown reduced the stability of NRF2, leading to a decrease in NRF2 mRNA level, suggesting that METTL3 is involved in the m6A modification of NRF2. Similarly, Ye et al. have also confirmed that NRF2 mRNA levels are regulated by m6A modification in hypopharyngeal squamous cell carcinoma (HPSCC). 31 Notably, RNA stability changes as “reader” proteins recognize m6A modification in the 3’-untranslated region (3’-UTR). 32 Our results indicated that METTL3 recruits YTHDF1, a reader protein, to recognize m6A modification sites in the 3’-UTR of NRF2, thereby promoting NRF2 translation; this, in turn, affects GPX4 expression and promotes ferroptosis in GBM cells, together propping up an METTL3/YTHDF1/NRF2/GPX4 axis in the regulation of ferroptosis in GBM. Our in vitro and in vivo experimental results indicated that EF24 significantly downregulates the protein and mRNA levels of METTL3, YTHDF1, NRF2, and GPX4. Furthermore, in the nude mouse orthotopic GBM model, EF24 markedly inhibits tumor growth and prolongs the survival of the mice. H&E staining showed that EF24 knockdown exerts no significant toxic damage on major organs. These findings suggest that targeting the METTL3/YTHDF1/NRF2/GPX4 signaling axis might serve as an effective and safe therapeutic strategy to combat GBM（Figure 9）. Further investigation is warranted to determine whether other key components in m6A modification, such as demethylases FTO and ALKBH5, are involved in the mechanism of action of EF24. Additionally, the synergistic effects of other ferroptosis inducers on EF24 should be explored. Figure 9 Mechanistic Investigation of EF24-Induced Ferroptosis in Glioma Cells Through the METTL3/YTHDF1/NRF2/GPX4 Axis EF24 can regulate m6A methylation to induce ferroptosis in glioma cells, with the METTL3/YTHDF1/NRF2/GPX4 axis participating in its regulatory mechanism. The underlying mechanism may involve EF24 downregulating METTL3 expression, thereby reducing the recognition capacity of the reader protein YTHDF1. This subsequently decreases the m6A modification level on NRF2 mRNA, inhibits NRF2 expression, and consequently downregulates GPX4 expression. These combined effects promote ferroptosis in glioma cells and ultimately exert antitumor activity. 5. Conclusion EF24 can regulate m6A methylation to induce ferroptosis in GBM cells. This mechanism is driven by an METTL3/YTHDF1/NRF2/GPX4 axis, in which EF24 mediates METTL3 expression to prevent m6A-modified mRNA from being recognized by the reader protein YTHDF1, thus promoting NRF2 translation. These findings provide a theoretical basis and potential drug targets for developing RNA epigenetics-based therapies for GBM. Conflicts of Interest The authors declare they have no competing interests. Acknowledgments Liaoning Provincial Department of Education, LJKMZ20221278, Study on the Role and Mechanism of Curcumin Analogs in Regulating m6A Methylation-Mediated Ferroptosis in the Occurrence and Progression of Glioma. Authors’ contributions Yang Yang made substantial contributions to the conception and writing.Liu Hao edited the manuscript.Zhou Jianxu and Yu Jingjie assisted in the animal experiments. Li Ge and Qu Nini resolved the major comments.Piao Hao Zhe confirmed the authenticity of all the raw data. All authors read and approved the final manuscript. The study was reviewed and approved by the Animal Ethics Committee of Dalian Medical University (number AEE23105), and all animal experiments were conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. References [1]Huang L, Zhang J, Gong F, et al. Identification and validation of ferroptosis-related lncRNA signatures as a novel prognostic model for glioma. Front Genet. 2022 Sep 20;13:927142. [2]Gómez Vecchio T, Corell A, Buvarp D, et al. Classification of Adverse Events Following Surgery in Patients With Diffuse Lower-Grade Gliomas. Front Oncol. 2021 Dec 21;11:792878. 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Authors Affiliations YANG YANG The First Affiliated Hospital of Dalian Medical University View all articles by this author Zhou Jianxu The First Affiliated Hospital of Dalian Medical University View all articles by this author Liu Hao Liaoning University of Traditional Chinese Medicine Affiliated Hospital View all articles by this author Qu Nini Liaoning University of Traditional Chinese Medicine Affiliated Hospital View all articles by this author Li Ge Dalian Hospital of Traditional Chinese Medicine View all articles by this author Yu Jingjie Dalian Hospital of Traditional Chinese Medicine View all articles by this author Piao Haozhe [email protected] Liaoning Cancer Hospital and Institute View all articles by this author Metrics & Citations Metrics Article Usage 314 views 168 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation YANG YANG, Zhou Jianxu, Liu Hao, et al. EF24 targets METTL3 to reprogram m6A methylation and induce ferroptosis: an epitranscriptomic mechanism with therapeutical potential for glioma. Authorea . 03 July 2025. DOI: https://doi.org/10.22541/au.175154669.93436266/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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