O-GlcNAcylation with ubiquitination stabilizes METTL3, promoting HMGB1 degradation to inhibit ferroptosis and enhance gemcitabine resistance in pancreatic cancer

O-GlcNAcylation with ubiquitination stabilizes METTL3, promoting HMGB1 degradation to inhibit ferroptosis and enhance gemcitabine resistance in pancreatic cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article O-GlcNAcylation with ubiquitination stabilizes METTL3, promoting HMGB1 degradation to inhibit ferroptosis and enhance gemcitabine resistance in pancreatic cancer qiuhong Wang, Dong Wei, Chunman Li, Xiawei Yang, Kun Su, Tao Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5606582/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Pancreatic cancer is highly lethal, and METTL3 plays a crucial role in m6A regulation. Although ferroptosis is important in cancer therapy, the regulatory mechanism of METTL3 in pancreatic cancer and its potential for regulating ferroptosis as a treatment remain unclear. Methods: The relationship between METTL3 and gemcitabine resistance and ferroptosis was investigated through in vitro and in vivo functional gain and loss experiments, while the target genes of METTL3 were identified using siRNA and pharmacological inhibitors to analyze the impact of O-GlcNAcylation on METTL3's stability and ubiquitination modification. Results: We found that O-GlcNAcylation interferes with ubiquitination-mediated regulation of METTL3 stability in pancreatic cancer cells, further clarifying its O-GlcNAcylation site and ubiquitination modification type. METTL3 regulates ferroptosis and Gemcitabine resistance by promoting HMGB1 degradation in a manner dependent on m6A-YTHDF2. Conclusion: Our study findings clearly demonstrate that METTL3 promotes pancreatic cancer cell proliferation and drug resistance to gemcitabine. This highlights the role of O-GlcNAcylation in stabilizing METTL3 expression and degrading HMGB1 through a m6A-YTHDF2-dependent mechanism, thereby inhibiting ferroptosis in pancreatic cancer cells. Furthermore, targeting HMGB1 while coordinating gemcitabine and RSL3 significantly suppresses pancreatic cancer tumor growth. These results provide valuable insights for the treatment of pancreatic cancer. Health sciences/Oncology/Surgical oncology Biological sciences/Cancer/Gastrointestinal cancer/Pancreatic cancer METTL3 Pancreatic cancer HMGB1 O-GlcNAcylation Gemcitabine resistance Ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Pancreatic cancer is a malignant tumor related to the gastrointestinal system 1 , typically presenting at an advanced metastatic stage, rendering it unresectable and exhibiting limited response to chemotherapy 2 . Surgical resection is not beneficial for over 80% of patients 3 , as those who undergo radical resection face a high risk of recurrence 4 . In recent years, there has been a global increase in the prevalence and mortality rates of pancreatic cancer, with only a 12.8% 5-year survival rate ( https://seer.cancer.gov/ ) observed. Gemcitabine (GEM)-based combined chemotherapy strategies remain the preferred treatment for advanced pancreatic cancer 5 . However, both GEM monotherapy and GEM combined with chemotherapy have shown very limited efficacy in improving the survival rate of patients with advanced PDAC due to GEM resistance 6 . Therefore, overcoming GEM resistance and identifying new targeted molecules against GEM are key challenges in the treatment of pancreatic cancer. Ferroptosis is an iron-dependent form of cell death characterized by excessive lipid peroxidation 7 . It primarily manifests as mitochondrial abnormalities, iron accumulation, and lipid peroxidation leading to plasma membrane rupture 8 . Recent studies have demonstrated the association between ferroptosis and GEM chemotherapy resistance in various tumor types, including pancreatic cancer 5 . Specific investigations have revealed that FBW7 enhances the cytotoxic effect of gemcitabine by activating both ferroptosis and apoptosis 9 , while SLC38A5 regulates ferroptosis to overcome gemcitabine resistance in pancreatic cancer 10 .In recent years, numerous studies have highlighted the role of HMGB1 in regulating cell ferroptosis 11,12 . In pancreatic cancer specifically, there are reports suggesting that targeting the MCP-GPX4/HMGB1 axis effectively triggers immunogenic ferroptosis in pancreatic ductal adenocarcinoma 13 . However, whether HMGB1 can be targeted to inhibit GEM resistance and promote ferroptosis for inhibiting the development of pancreatic cancer remains unexplored. We observed that the binding free energy between HMGB1 and GEM in the protein-molecule docking model is less than zero, indicating a potential interaction between HMGB1 and GEM as a target molecule. This suggests that targeting HMGB1 may promote ferroptosis in pancreatic cancer cells while inhibiting GEM resistance and preventing further progression of this disease. O-GlcNAcylation is a reversible posttranslational modification of proteins 14 , resulting from glucose metabolism via the hexosamine biosynthesis pathway (HBP), which integrates glucose, amino acids, fatty acids, and nucleotides 15 . The regulation of O-GlcNAcylation involves two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) 16 . Since its discovery, O-GlcNAcylation has been implicated in various cellular functions including signal transduction, protein localization and stability, transcriptional control, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation of the O-GlcNAc cycle is associated with the progression of diverse diseases such as diabetes mellitus and its complications as well as cancer and cardiovascular/neurodegenerative disorders 17 . In pancreatic cancer specifically, emerging evidence suggests that alterations in O-GlcNAcylation impact disease progression through modulation of different substrates. For instance: promotion of pancreatic tumor growth by regulating malate dehydrogenase 1 through O-GlcNAcylation has been reported 18 ; facilitation of ferroptosis in mesenchymal pancreatic cancer cells via ZEB1's O-GlcNAcylation has also been observed 19 ; additionally,O-GlcNAcylation-mediated stabilization of SIRT7 promotes pancreatic cancer progression by disrupting the SIRT7-REGγ interaction 20 . Therefore,it is imperative to identify novel substrate proteins for targeted regulation of pancreatic cancer progression. M6A is the most prevalent epigenetic modification in eukaryotic cells and governs diverse biological processes 21 . The three primary regulatory factors of M6A epigenetic alterations are the M6A methyltransferase (writer), M6A demethylase (eraser), and M6A recognition protein (reader) 22,23 . METTL3 functions as an S-adenosylmethionine (SAM) binding protein and acts as a catalyst by utilizing its internal SAM-binding domain to transfer methyl from SAM to the adenine base of RNA, resulting in the production of S-adenosylhomocysteine (SAH) 21 . Emerging evidence suggests that METTL3 plays a crucial role in cancer development, acting either as an oncogene or tumor suppressor gene 24 , for instance: METTL3-mediated m6A modification of HDGF mRNA promotes gastric cancer progression 25 . Wang et al. also reported overexpression of METTL3 in breast cancer and identified BCL2 as a target of METTL3. They demonstrated that elevated m6A modification in Bcl-2 mRNA promoted its translation and ultimately facilitated cancer cell proliferation 26 . METTL3 facilitates m6A modification of suppressor of cytokine signaling 2 (SOCS2) and promotes its mRNA degradation through a YTHDF2-dependent pathway, thereby regulating the progression of liver cancer 27 . Studies have demonstrated that elevated levels of METTL3 expression are correlated with advanced pathological stages in PDAC 28 , as well as resistance to chemotherapy and radiotherapy in pancreatic cancer cells 29 . However, the underlying mechanisms remain elusive. Therefore, comprehending the molecular mechanisms and downstream targets governed by METTL3 in pancreatic cancer regulation may offer novel avenues for the treatment and diagnosis of this disease. In this study, we investigated the molecular mechanism underlying the role of METTL3, an M6A WRITER, in pancreatic cancer development and its impact on gemcitabine sensitivity. Our findings demonstrate that METTL3 interacts with OGT and undergoes O-GlcNAcylation, thereby elucidating the specific modification site involved in O-GlcNAcylation of METTL3. This post-translational modification stabilizes METTL3 expression by enhancing its interaction with EIF3H, a deubiquitinating enzyme. Additionally, we discovered that METTL3 promotes HMGB1 degradation through a m6A-YTHDF2-dependent pathway, inhibits ferroptosis in pancreatic cancer cells, and confers resistance to gemcitabine treatment. Overall, our study uncovers a novel regulatory axis involving OGT-METTL3-HMGB1 that governs pancreatic cancer development and highlights the potential therapeutic strategy targeting ferroptosis and gemcitabine. METHODS Cell lines ， cell cultures and transfer The PANC1 and BXPC3 cell lines, along with the HEK293T cell line, were acquired from a Chinese cell bank situated in Shanghai. These cellular cultures were maintained using Dulbecco‘s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), along with penicillin at a concentration of 100 U/ml and streptomycin at a concentration of 100 μg/ml. The incubation process involved keeping the cells at a temperature of 37℃ within an environment saturated with humidity and containing approximately 5% CO 2 . In order to achieve transient expression of plasmids within HEK293T cells, we employed PEI transfection solution based on findings reported by Zhou et al 30 . When introducing plasmids into various cancerous cell lines, Lipofectamine™️3000 manufactured by Invitrogen was utilized according to guidelines provided by its manufacturer. The siRNA sequences ：siYTHDF2：5′-GCACAGAAGTTGCAAGCAA -3′; siOGT：5′-GCCUGAUAGAUCUGGCAAUTT-3′; siEIF3H: 5ʹ-GCAACTCTTGGAAGAAATATA-3ʹ. PugNAc (ab144670) was purchased from Abcam； Cycloheximide (S7418) was purchased from Selleck. Actinomycin D(50-76-0) was purchased from MCE. Clinical samples The tumor tissue and matched non-tumor tissue samples of pancreatic cancer were obtained from 7 patients who underwent surgery at The Second Affiliated Hospital of Kunming Medical University. All patients provided informed consent and did not receive any preoperative chemotherapy or radiotherapy. This study was approved by the The Second Affiliated Hospital of Kunming Medical University Ethics Review Board. Cell Viability Assay The Cell Counting Kit-8 (CCK-8) (B34302, Bimake) was used according to the manufacturer's instructions. Briefly, cells were seeded in 96-well plates at a density of 1 × 104 cells per well. For the gemcitabine treatment group, cells were treated with various concentrations of gemcitabine for a specified duration of 24 hours. Subsequently, CCK-8 reagent (10 μl) was added to each well and incubated in a 5% CO2 incubator at 37°C for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Quantitative real time-PCR Total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA). The cDNA synthesis was performed by reverse transcribing the RNA with a reverse transcription kit (Bio-Bio Engineering (Dalian) Co., Ltd.). Real-time fluorescent quantitative PCR was conducted using the SYBR-Green PCR Master Mix kit (TOYOBO, Japan). Please refer to supplementary table 1 for the sequences. Western blot Cells were harvested and disrupted using a solution containing SDS. The resulting proteins were separated by electrophoresis on an SDS-PAGE gel and subsequently transferred to a PVDF membrane. The PVDF membrane was then blocked with non-fat milk (5%). Specific primary antibodies were applied to the PVDF membrane for incubation, followed by secondary antibody treatment. Detection of proteins was achieved through chemiluminescence analysis. The following is the antibody information: anti-Flag (F1804) antibodies were purchased from Sigma (MO, USA); anti-β-actin (#4970), anti-O-GlcNAc (#9875) and anti-HMGB1(#3935)were obtained from Cell Signaling Technology (MA, USA), anti-eIF3h (ab60942) was obtained from Abcam (Cambridge, UK). anti-HA (sc-57592) antibodies were purchased from Santa Cruz biotechnology (TX, USA); anti-Myc (60003-2-Ig), anti- METTL3 （15073-1-AP）and anti-YTHDF2(24744-1-AP) were purchased from Proteintech. Cycloheximide treatment The corresponding cells were seeded onto 6-well plates and subjected to treatment with cycloheximide (20 mg/mL) for 0, 4, 8, and 12 hours prior to collection. Total proteins were extracted and subsequently analyzed by Western blotting using the respective antibodies. mRNA stability analysis Actinomycin D, a transcriptional inhibitor commonly employed for RNA stability detection, was administered to transfected cells at a concentration of 5 μg/ml. Subsequently, the cells were collected at designated time intervals and total RNA was extracted using TRIzol reagent. The relative expression levels of HMGB1 mRNA were assessed via qRT-PCR analysis. ME-ELISA The EpiQuik TM m6A RNA Methylation Quantification Kit (Colorimetric) (Epigentek, USA) is utilized for the colorimetric determination of total m6A levels in pancreatic cancer cells. Specifically, 200 ng of RNA is immobilized onto capture antibodies in each well for subsequent detection. Following multiple incubation steps, the m6A content is quantified using a colorimetric method at 450 nm and calculated based on a standard curve. MERIP-qPCR The MERIP-qPCR procedure was conducted following the guidelines provided by the EpiQuik ™ CUT&RUN m6A RNA Enrichment (MeRIP) Kit. In brief, an immunocapture solution was prepared by combining reagents in 0.2 ml PCR tubes and rotating them at room temperature for 90 minutes. Each tube received 10 μl of NDE (Nuclear Digestion Enhancer) and 2 μl of CEM (Cleavage Enzyme Mix), followed by a 4-minute incubation at room temperature. The tubes were then placed on a magnetic device until the solution became clear, which took approximately 2 minutes. After discarding the supernatant, samples underwent three washes with 150 μl of WB (Wash Buffer), followed by one wash with 150 μl of PDB (Protein Digestion Buffer). Subsequently, samples were mixed with 20 μl of Protein Digestion Solution and incubated at a temperature of 55 ◦C for a duration of 15 minutes using a thermocycler without a heated lid. RPS (RNA Purification Solution) and absolute ethanol were applied to the samples to resuspend and cleanse the RNA Binding Beads through vortexing. The resuspended beads were then subjected to treatment with Elution Buffer totaling13 μl, allowing for an incubation period at room temperature lasting for5 minutes to release RNA from the beads.Finally,13μlof each sample was transferred into new0.2ml PCR tubes either for immediate use or storage at -20℃ RIP‑qPCR The endogenous RNA was captured in the nucleus or cytoplasm through antibody or epitope labeling, followed by isolation of RNA-binding proteins from the bound RNA using immunoprecipitation. Cells were crosslinked with 1% formaldehyde and treated with RIPA buffer containing 150 mM NaCl, RNase, and protease inhibitors. Subsequently, cell lysis was performed using a solution consisting of 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40, 1 mM EDTA, and 50 mM Tris (pH 8.0) for a duration of 30 minutes before centrifugation to collect the precipitates. The supernatant was then incubated four times with primary antibodies against METTL3 and YTHDF2 (or corresponding IgG antibodies), followed by addition of protein A/G glycosylated microspheres which were shaken for two hours. After washing the cells three times with RIPA buffer, RNA extraction was carried out following crosslinking procedures. Finally, quantitative RT-PCR was employed to detect the extracted RNA. RNA pull‑down assays The initial step involved the utilization of the MEGAscript T7 Transcription Kit from Thermo Scientific to transcribe the RNA. Subsequently, we employed the Pierce RNA 3′ End Desthiobiotinylation Kit (20, 163, Thermo Scientific) to label the ends of the amplified RNA with desthiobiotin. Lastly, we conducted RNA pulldown experiments using the Pierce Magnetic RNA–Protein Pull-Down Kit (20, 164, Thermo Scientific). Specifically, a mixture was prepared by combining 2 mg of protein lysates, 50 pmol of biotinylated RNAs, and 50 µL of streptavidin beads. Following three washing cycles and an incubation period, immunoblotting analysis was performed after subjecting the streptavidin beads to boiling. Luciferase reporter assay The regions of HMGB1 mRNA containing the methylation sites of METTL3 were cloned into a pGL3 plasmid. The mutated sequence was synthesized by GenePharma, located in Shanghai, China. To quantify luciferase activity, we utilized the dual-luciferase reporter assay system from Promega based in the United States. Using a GloMax 20/20 Luminometer also provided by Promega, we determined the relative luciferase activity as indicated by the ratio between firefly luciferase activity and Renilla luciferase activity. Immunohistochemical (IHC) staining For clinical specimens and mouse xenograft samples, paraffin embedding was carried out using the R.T.U. Vectastain Kit (from Vector Laboratories). Immunohistochemical staining was performed according to the instructions provided with the immunohistochemistry kit. All stainings were evaluated using quantitative imaging methods, where the percentage and intensity of immunostaining were recorded. The H-score was calculated using the following formula: H-score = Σ (PI × I) = (Percentage of cells with weak intensity × 1) + (Percentage of cells with moderate intensity × 2) + (Percentage of cells with strong intensity × 3) Here, PI represents the percentage of positively stained cells among all cells, and I denotes the staining intensity. Ki-67 (Proteintech: 27309-1-AP, China); anti-CD133 (Proteintech: 66666-1-Ig, China) Plasmid Construction and Lentiviral infection All plasmids were designed to clone the corresponding cDNA into the corresponding expression vector to produce the desired protein. For shRNA and PCDH vectors, the culture supernatant of HEK 293T cells was collected 48-72 hours after transfection for virus preparation and target cells were infected with 70% infection efficiency. PCDH lentivirus was used to construct METTL3 wild-type (WT) overexpression cell lines, while lentiCRISPR method was used to generate METTL3 knockout (KO) cell lines. In brief, we used BsmBI enzyme to linearize the lentiviral CRISPR vector and constructed the guide RNA (shRNA) into the lentiCRISPR V2 lentiviral expression vector. The shMETTL3 sequence: 5'-GCACTTGGATCTACGGAATCC-3'. Proliferation Assay The cellular proliferation was assessed using the EdU kit (provided by KeyGen Biotech Co., Ltd.) to quantify the EdU incorporation. After 48 hours of transfection, cells were seeded at a density of 2 × 105 cells per well in a 6-well plate and exposed to 10 nM EdU for a duration of 12 hours. Subsequently, the cells were fixed and permeabilized with 0.5% Triton X-100 for a period of 20 minutes. Following this, the Click-iT reaction cocktail was introduced, and the cells were incubated under dark conditions for approximately half an hour. After two washes with PBS, DAPI dye was applied as a counterstain for about ten minutes in darkness before observing them using fluorescence microscopy. Sphere-forming assay The PC cells were cultured in serum-free DMEM/F12 medium supplemented with 2% B-27 (Gibco, USA, #17504044), heparin (4 μg/ml), epidermal growth factor (20 ng/ml) (R&D, USA, #236-EG-200), and fibroblast growth factor (20 ng/ml) (PEPROTECH, USA, #AF-100-18C). DMEM/F12 medium with varying glucose concentrations was prepared by combining equal volumes of low-glucose DMEM medium (Gibco, USA) and F12 medium (Gibco, USA) containing d-glucose from Aladdin Industrial Corporation (#G116304). The cells were seeded at a density of 500 cells per well in ultra-low adhesion 6-well plates. After 7 days of incubation, the cultures were examined for sphericity using a phase contrast optical microscope. Flow cytometry To perform staining, a total of 5×10 5 cells were seeded into a U-shaped 96-well plate and incubated with 5 µl of each antibody at a temperature of 4°C for a duration of 30 minutes. Following PBS washing, the cells were collected by centrifugation at a gravity force of 1000 g for 5 minutes. Subsequently, the cells were resuspended in 300 µl of PBS and subjected to flow cytometry analysis. The CD133/1-PE antibody was procured from MiltenyiBiotec located in Bergisch Gladbach, Germany. Mitochondrial superoxides measurements The MitoSOX™ Red mitochondrial superoxide indicator for live-cell imaging (Invitrogen) was utilized to measure the accumulation of mitochondrial superoxide. The manufacturer's protocol was followed for this purpose. Briefly, adherent cells were cultured on glass coverslips in a six-well plate. After the specified treatments, a 5 μM MitoSOX™ working solution (1 ml) was added to the cells on the coverslips. Subsequently, incubation of the cells at 37°C in darkness took place for 10 minutes. Following this, gentle washing with warm buffer was performed three times. Mounting of coverslips in warm buffer facilitated imaging using a confocal microscope. Determination of intracellular ROS, malondialdehyde (MDA), and glutathione (GSH) The CellROX™ Deep Green Reagent (Invitrogen, C10444) is employed in the experimental procedure to observe fluorescence microscopy and detect intracellular levels of ROS. Briefly, prior to treatment, samples are exposed to a 5 μM solution of the reagent for a duration of 30 minutes. Subsequently, after a post-treatment period lasting 0.5 hours, cells are gathered for measurements of fluorescence intensity. The evaluation of Malondialdehyde (MDA) and Glutathione (GSH) production within the cells is conducted using MDA Detection Kit and GSH Assay Kit respectively, which are provided by Solarbio Science & Technology Co., Ltd., Beijing, China. Mitochondrial membrane potential measurements Tetramethylrhodamine Ethyl Ester (TMRE) (manufactured by Beyotime) is a commonly employed fluorescent dye for labeling mitochondria in viable cells. As per the provided guidelines, an appropriate quantity of TMRE buffer is introduced to the cells, which are then incubated at 37°C in a light-restricted environment for a duration of 20 minutes. Following this, the cells are rinsed with serum-free medium and subsequently observed using a fluorescence microscope. Intracellular iron assay The concentration of ferrous ions within the cell was assessed by utilizing FerroOrange dye (#F374, Dojindo Laboratories). Cells were cultured and subjected to specific treatments. Subsequently, a diluted solution (1:2000, v/v) of FerroOrange Orange dye was added to the cells and incubated at 37°C for 30 minutes. Fluorescent images were captured using a confocal microscope. Immunoprecipitation (IP) and Co-immunoprecipitation (co-IP) Cells were disrupted in Pierce IP buffer (Thermo Fisher) supplemented with protease and phosphatase inhibitors (Sigma). Following incubation with specified antibodies, the lysates were combined with protein A/G agarose beads (Thermo Fisher). For proteins carrying a tag, we employed immunomagnetic beads conjugated to anti-Flag/anti-HA antibody. Subsequent to immunoprecipitation, protein A/G agarose or magnetic beads underwent three washes using TBST (0.1% Tween-20, 150 mM NaCl, 10 mM Tris-HCl pH7.5), followed by elution in SDS lysis buffer (100 mM NaCl, 1% SDS, 50 mM Tris-HCl pH 7.5) for western blot analysis. In vivo ubiquitination assay In ubiquitination experiments, cells were transiently transfected with the specified plasmids for 36 hours, followed by treatment with 20 μM MG132 for an additional 8 hours. The cells were then harvested. One-fifth of the cells were reserved for direct immunoblotting, while the remainder were processed for denaturing co-immunoprecipitation. The cells were lysed in denaturing buffer (described by Liu et al., 2023), and the lysates were incubated overnight HA-beads (Millipore; IP0010) at 4°C. Subsequently, the beads were washed with buffers and Buffer C. Finally, the immunocomplexes were eluted using elution buffer, and the samples were analyzed by immunoblotting with indicated antibodies as detailed by Liu et al 31 . Xenograft Model our-week-old nude mice were purchased from Cavens, Changzhou, China. The nude mice were randomly allocated into 5 groups without any specific selection criteria. The mice were raised under specific pathogen-free conditions. Pancreatic cells (1 × 10 6 ) stably expressing METTL3 and HMGB1 were implanted into the right flank of each mouse and allowed to grow, with 6 mice in each group (n = 6). Six days after cancer cell injection, gemcitabine (120 mg/kg, Sigma-Aldrich) was administered weekly for the gemcitabine treatment cohort, while RSL3 (5 mg/kg, Sigma-Aldrich) was injected once daily for the combined gemcitabine and RSL3 treatment group. Both gemcitabine and RSL3 were administered via intraperitoneal injection. This treatment regimen continued until the final observation week. The animal experimental protocol was approved by the Animal Ethics Committee of the Animal Care and Use Committee of the Ethical Institution of The Second Affiliated Hospital of Kunming Medical University. The investigators did not blind the animal experiments. Bioinformatical analysis The survival curve of METTL3 in pancreatic cancer was obtained from the database https://smuonco.shinyapps.io/PanCanSurvPlot/; METTL3 differential genes and METTL3 peak genes were obtained from GSE146806 and GSE132306; METTL3 targeted genes were downloaded from the database http://rm2target.canceromics.org/#/home, and METTL3 differential genes in pancreatic cancer were obtained from UALCAN (uab.edu); ferroptosis-related genes were obtained from FerrDb (zhounan.org); the expression of related proteins such as METTL3 and YTHDF2 in pancreatic cancer was downloaded from Proteomic Data Commons (cancer.gov); Welcome to SRAMP, an online m6A site predictor (cuilab.cn) predicted HMGB1 m6A modification sites; the docking model of HMGB1 and gemcitabine was analyzed by autodockvina and visualized by Pymol. Statistical analysis Data were collected from a minimum of three independent experiments. Statistical analysis was conducted using Prism software (GraphPad Prism 9.0.0). Unless otherwise stated, all data are presented as mean ± standard deviation. Paired or unpaired Student's t-test (two-tailed) was employed for experiments involving two groups only. One-way ANOVA with multiple comparisons was utilized for experiments with more than two groups. Two-way ANOVA with multiple comparisons was applied for comparing four or more groups in a two-factor experiment. The level of statistical significance was set at *p<0.05, ** p<0.01 to determine significant differences among the experimental groups. RESULTS METTL3 is upregulated in pancreatic cancer and promotes pancreatic cancer cell proliferation, stemness, and gemcitabine resistance To investigate the impact of METTL3 expression on the prognosis of pancreatic cancer patients, we utilized the PanCanSurvPlot database and observed that patients with high METTL3 expression exhibited a poor prognosis (Fig. 1 A). We collected multiple samples of pancreatic cancer tissues along with adjacent normal tissues and detected an upregulation in METTL3 expression in tumor tissues through qPCR analysis (Fig S1 A). Furthermore, immunohistochemistry and Western blot analyses confirmed a significant upregulation of METTL3 expression in tumor tissues compared to normal tissues (Fig. 1 B, C). In order to clarify the role of METTL3 in the progression of pancreatic cancer, we established stable knockdown and overexpression models of METTL3 in two pancreatic cancer cell lines: PACN1 and BXCP3 (Fig S1 B). Utilizing the CCK8 assay, we discovered that overexpression of METTL3 promoted proliferation in both PACN1 and BXCP3 cells, while knockdown of METTL3 inhibited their growth (Fig S1 C, D). Furthermore, we observed a decrease in the number of EdU-positive cells in PACN1 and BXCP3 upon knockdown of METTL3 compared to the control group (Fig. 1 D). Gemcitabine is currently considered as the first-line treatment for pancreatic cancer; however, resistance to gemcitabine has emerged as a major obstacle. Interestingly, our findings indicate that lower levels of METTL3 exhibit greater sensitivity to gemcitabine compared to higher levels of METTL3 (Fig. 1 E, F). Given that cell pluripotency has been linked to GEM resistance 32 , we further investigated the impact of METTL3 on pancreatic cancer stemness. Initially, we conducted cell sphere formation experiments and observed a significant reduction in both volume of spheres formed by PANC1 and BXPC3 cells following METTL3 knockout (Fig. 1 G; Fig S1 E). Additionally, flow cytometry analysis revealed a notable decrease in the percentage of CD133 + cells - a marker for stemness - upon METTL3 knockout in PANC1 and BXPC3 cells (Fig. 1 H; Fig S1 F). The inhibition of ferroptosis in pancreatic cancer cells is mediated by METTL3 To further elucidate the molecular mechanism underlying METTL3-mediated regulation of pancreatic cancer progression, we retrieved the GSE146806 and GSE132306 datasets from the GEO database. Subsequently, a KEGG analysis was performed on the overlapping genes between the top 2,000 differentially expressed genes identified in GSE146806 and the genes corresponding to METTL3 binding RNA peaks in GSE13,306. Our findings revealed that METTL3 is involved in regulating ferroptosis (Fig S2 A). Consequently, knockdown of METTL3 in stable knockout and stable overexpression models of PANC1 and BXPC3 led to an elevation in mitochondrial superoxide levels (Fig. 2 A, Fig S3 A), increased MDA production (Fig. 2 B, Fig S3 B), reduced GSH levels (Fig. 2 C, Fig S3 C), accompanied by augmented ROS levels (Fig. 2 D, Fig S3 D) and iron ion concentrations (Fig. 2 E, Fig S3 E), as well as decreased mitochondrial membrane potential levels (Fig. 2 F, Fig S3 F). Conversely, overexpression of METTL resulted in an opposite phenotype. Collectively, these results indicate that METTL inhibits cell ferroptosis and consequently promotes pancreatic cancer progression. HMGB1 was identified as a downstream target of METTL3 As METTL3 is an M6A methyltransferase, we firstly detected the effect of METTL3 on the overall m6A level in PANC1 and BXPC3 cells. The results showed that METTL3 significantly increased the overall m6A level (Fig. 3 A, Fig S4 A). The above experiments have shown that METTL3 regulates ferroptosis in pancreatic cancer cells. In order to determine the downstream ferroptosis target regulated by METTL3, we used a Venn diagram showing differential genes obtained from GSE146806 analysis, correlation genes of METTL3 in pancreatic cancer obtained from UALCAN, target genes from RM2Target database, and the genes corresponding to METTL3 binding RNA peaks in GSE13306 and ferroptosis-related genes to obtain HMGB1 (Fig. 3 B). The RIP-qPCR assay was employed to validate the binding of METTL3 in PDAC cells with either knockdown or overexpression. Overexpression of METTL3 led to an increased enrichment of HMGB1, whereas knockdown of METTL3 resulted in a significant decrease (Fig. 3 C, Fig S4 B). RNA pull-down analysis demonstrated the direct interaction between full-length HMGB1 mRNA and the METTL3 protein (Fig. 3 D). MeRIP-qPCR results revealed that the presence of METTL3 directly enhanced m6A modification on HMGB1 mRNA in PANC1 and BXPC3 cells (Fig S4 C, D). Furthermore, Western blotting showed that depletion of METTL3 elevated HMGB1 protein levels, while overexpression of METTL3 reduced them (Fig S4 E), which was further confirmed by qPCR analysis (Fig S4 F). Additionally, actinomycin D treatment was performed to assess the impact of METTL3 on HMGB1 mRNA stability, demonstrating that knockdown of METTL3 resulted in increased stability for HMGB1 mRNA (Fig. 3 E, F). We referred to the SRMAP database ( http://www.cuilab.cn/sramp ) to determine the m6A sites, and found two highly confident m6A sites in the 3' UTR. Next, we conducted a luciferase assay by constructing the 3' UTR of HMGB1 (Fig S4 G) to evaluate the effect of METTL3 binding on transcription. In the mutant group, the adenine nucleotide at the m6A site was replaced with cytosine to eliminate the m6A modification. The relative luciferase activity (Firefly/ Renilla ratio) ) showed that METTL3 silencing enhanced the transcriptional activity of the 3' UTR of HMGB1. It is noteworthy that the mutation at position 1535 eliminated the effect of METTL3 silencing, while the mutation at position 791 did not (Fig. 3 G). The MeRIP-qPCR results further confirmed that position 1535 mediated the m6A methylation of HMGB1 mRNA (Fig. 3 H, Fig S4 H) and the stability of HMGB1 mRNA (Fig. 3 I, Fig S4 I). YTHDF2 mediates HMGB1 mRNA expression in an m6A-dependent manner The dynamic and reversible regulation of m6A modification is determined by the interaction between m6A writers and erasers. However, different downstream biological functions necessitate the recognition of m6A by distinct readers, including the regulation of m6A-modified transcripts. Stability analysis of HMGB1 mRNA revealed that METTL3 deletion (Fig. 3 E, F) and mutation at the m6A site on HMGB1 mRNA (Fig. 3 I, Fig S4 I) both enhanced mRNA stability. Previous studies have reported that YTHDF2 recognizes m6A modification to promote degradation of its target gene mRNA 33 . Through RNA pull-down experiments and western blotting, we identified YTHDF2 as an m6A reader for HMGB1 mRNA in PANC1 and BXPC3 cells (Fig. 4 A). qPCR experiments demonstrated a negative correlation between YTHDF2 expression and HMGB1 mRNA expression (Fig. 4 B), which was further confirmed by western blotting showing increased levels of HMGB1 protein upon knocking down YTHDF2 (Fig. 4 C, D). Analysis of protein expression data from the Proteomic Data Commons database revealed a negative correlation between YTHDF2 and HMGB1 protein expression in pancreatic cancer samples (Fig. 4 E). Additionally, RIP-qPCR experiments showed that knocking down YTHDF2 led to increased levels of HMGB1 mRNA (Fig. 4 F). Relative luciferase activity assays indicated that silencing YTHDF2 enhanced luciferase activity associated with HMGB1, while mutation at site 1535 abolished this effect caused by YTHDF2 silencing (Fig. 4 G), providing further evidence for the role of knocking down YTHDF2 in enhancing stability of HMGB1 mRNA in PANC-1 and BXPC-3 cells (Fig. 4 H). Our findings suggest a mechanistic control exerted by YTHDF2 on stability and expression levels of HMGB1 mRNA through an m6A-dependent manner. Inhibition of ferroptosis by METTL3 and gemcitabine resistance are mitigated by HMGB1 Our previous studies have demonstrated that METTL3 inhibits ferroptosis and induces resistance to gemcitabine. HMGB1, a downstream target of METTL3 in the context of ferroptosis, the association between ferroptosis and gemcitabine resistance has been extensively documented in the literature. To investigate whether HMGB1 can regulate the sensitivity of pancreatic cancer cells to gemcitabine, we initially employed autodock vina software for analyzing the free energy between HMGB1 and gemcitabine molecules. The complexing free energy between HMGB1 and GEM was found to be -6.1 kcal/mol. Additionally, we visualized the docking model using pymol software (Fig S5 A), suggesting that HMGB1 may serve as a downstream molecular target of GEM. We evaluated the impact of HMGB1 on GEM sensitivity in PANC1 and BXPC3 cells overexpressing METTL3. Our findings revealed that HMGB1 could enhance the sensitivity of METTL3-overexpressing cells towards GEM treatment (Fig. 5 A, B). Similarly, HMGB1 could mitigate the effect of METTL3 on cell stemness by reducing sphere-forming cell diamater (Fig. 5 C) and CD133 + cell populations (Fig S5 B). Subsequently, we investigated whether HMGB1 could alleviate the inhibition of ferroptosis caused by METTL3 in pancreatic cancer cells. For non-transfected cells, transfection with HMGB significantly reduced ROS levels (Fig. 5 D) and MDA levels (Fig. 5 E), while upregulating GSH levels (Fig. 5 E). These results suggest that through alleviating ferroptosis inhibition and gemcitabine resistance induced by METTL3 overexpression, HMGB contributes to cellular responses in pancreatic cancer cells. To explore synergistic effects among gemcitabine treatment, ferroptosis induction, and HMBG expression on cell proliferation rates; we first assessed cell viability revealing that overexpression of HMBG combined with treatment involving both gemcitabine and RSL3 significantly reduced cell viability rates (Fig. 5 F, G). The combined treatment of HMGB1 and GEM with RSL3 significantly suppressed tumor proliferation in in vivo experiments (Fig. 5 H, Fig S5 C, D), immunohistochemistry also confirmed this result (Fig. 5 I). These findings suggest that targeting HMGB1 in conjunction with GEM and ferroptosis represents a promising therapeutic strategy for pancreatic cancer. O-GlcNAcylation stabilizes METTL3 protein via enhancing the interaction between METTL3 and EIF3H Our previous studies have demonstrated that high expression of METTL3 is associated with a poor prognosis in pancreatic cancer and promotes the progression of pancreatic cancer cells. In order to identify new molecular mechanisms regulating the function of METTL3, we discovered its involvement in lipid metabolism, central carbon metabolism, amino acid metabolism, and nucleic acid metabolism (Fig S2 A). Additionally, O-GlcNAcylation integrates glucose, amino acids, fatty acids, and nucleic acid metabolism 15 . As shown in the UALCAN database, OGT also shows a significant upregulation of protein expression in pancreatic cancer (Fig S6 A). Therefore, we hypothesized whether METTL3 undergoes O-GlcNAcylation mediated by OGT (O-GlcNAc Transferase). Initially, we obtained the 3D structure of the METTL3-METTL14 complex and OGT dimer from the PDB database ( https://www.rcsb.org/ ), as well as the complex model of METTL3-METTL4 and OGT from Cluspro (Fig S7 A). The binding free energy analysis using https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver revealed that the binding free energy between METTL3-OGT is less than 0༈Fig S7 B༉, suggesting an interaction between them. Co-IP experiment confirmed this interaction between METTL3 and OGT (Fig. 6 A). Furthermore, while the K908A mutation in OGT eliminated its enzymatic function without affecting protein abundance according to previous research 34 , our study found that this mutant could still interact with METTL3. However, this mutant failed to induce O-GlcNAcylation of METTL3, suggesting that enzymatic activity of OGT is necessary for the process (Fig. 6 A). To determine the region where OGT and METTL3 interact, we constructed a truncated model of OGT (Fig S7 C). We found that, except for the δTPR region, METTL3 could bind to it (Fig S7 D), indicating that METTL3 interacts with the OGT-TPR region. Since O-GlcNAcylation usually affects protein stability, we downloaded the expression of METTL3 and OGT proteins in pancreatic cancer patients from the Proteomic Data Commons and plotted a correlation curve, which showed that OGT protein expression and METTL3 protein expression were positively correlated in pancreatic cancer (Fig S7 E). In PANC1 cells, we detected the expression of METTL3 and the overall O-GlcNAcylation by interfering with OGT and giving PugNAc to inhibit OGA, and found that after inhibiting OGT, the expression level of METTL3 protein was lower, while inhibiting OGA could enhance METTL3 protein expression, and the expression of METTL3 was significantly downregulated after O-GlcNAcylation inhibition (Fig. 6 B, C). However, O-GlcNAcylation did not affect the expression of METTL3 mRNA (Fig S7 F). Next, we detected the effect of O-GlcNAcylation on the stability of METTL3 protein. As shown in Fig. 6 D, knocking down OGT and inhibiting O-GlcNAcylation promoted the degradation of METTL3. The ubiquitin-proteasome pathway mediates 80%-85% of protein degradation 35 . O-GlcNAcylation can prevent the degradation of target proteins by reducing their ubiquitination, and the underlying mechanism is to recruit deubiquitinating enzymes to O-GlcNAcylated proteins 16 . The ubiquitination test also confirmed that knockdown of OGT and K908A mutations did enhance the level of METTL3 ubiquitination (Fig. 6 E), further clarifying that OGT mainly mediates polyubiquitination in a K48-dependent manner (Fig S7 G). Next, we explored the mechanism by which O-GlcNAcylation regulates the stability of METTL3. We downloaded the binding protein of METTL3 (PXD036899) from the proteomexchange.org database, downloaded the interacting proteins of METTL3 and all deubiquitinating enzymes from the databases https://thebiogrid.org/ and https://iuucd.biocuckoo.org/index.php respectively, and obtained the only overlapping protein EIF3H through the Venn diagram (Fig S7 H). We verified that there was no correlation between EIF3H mRNA and METTL3 mRNA in the GEPIA database (Fig S7 I), but a positive correlation at the protein level (Fig S7 J). The above data illustrate that EIF3H regulates the expression of METTL3 protein. The ubiquitination experiment demonstrated that knockdown of EIF3H significantly enhanced the ubiquitination of METTL3 (Fig. 6 F). Through Co-IP experimentation, we observed a binding interaction between EIF3H and METTL3(Fig. 6 G). By analyzing the binding free energy of EIF3 and METTL3, as well as EIF3 and METTL3-OGT complexes using Cluspro, we discovered that the binding free energy of EIF3 and METTL3-OGT complexes was lower compared to that of EIF3 and METTL3 within a similar interaction area. This suggests that OGT has the ability to enhance the binding affinity between METTL3 and EIF3 (Fig S6 B). The Co-IP results further confirmed this observation, demonstrating that the addition of OGT augmented the interaction between METTL3 and EIFH (Fig. 6 G). Similarly, PugNAc treatment also enhanced the binding between METTL3 and EIF3H (Fig. 6 H). We combined multiple databases ( https://services.healthtech.dtu.dk/services/NetOGlyc-4.0/;https://services.healthtech.dtu.dk/services/DictyOGlyc-1.1/ ༛ https://www.oglcnac.mcw.edu/ ༉ to predict that the O-GlcNAcylation modification site of METTL3 is S118, and this site is highly conserved in mice, rats and humans. We produced a mutant of METTL3 (S118A). The IP assay also showed significantly lower levels of O-GlcNAcylation on S118A compared to WT (Fig S7 K), indicating that S118 is the main site of O-GlcNAcylation of METTL3. Compared with WT, the S118A mutant promoted the degradation of METTL3 (Fig. 6 I), inhibited the binding of METTL3 and EIF3H (Fig. 6 J), and the ubiquitination experiment also proved that the S118A mutation significantly enhanced the ubiquitination modification of METTL3 compared with WT(Fig S7 L). The findings suggest that O-GlcNAcylation promotes the stability of METTL3 by enhancing the interaction between METTL3 and EIF3H. DISCUSSION More and more evidence suggests that M6A modification plays a crucial role in various biological processes, especially in tumor formation and cancer progression. As the catalytic subunit of the M6A writer, METTL3 has been found to collaborate with YTHDF2 and promote HCC progression by degrading the mRNA of SOCS2 27 . In pancreatic cancer, it has also been proven that METTL3 modulates ID2 by m6A methylation to regulate pancreatic cancer cell stemness 36 . Previous studies mainly focused on the role of METTL3 in mediating mRNA metabolism and tumor progression through its target gene m6A methylation regulation, however, the limited understanding of METTL3-specific functional regulation, especially in post-translational modification, still exists. Here, we first prove that METTL3 is highly expressed in pancreatic cancer and inhibits ferroptosis-induced gemcitabine resistance, and HMGB1 is identified as a downstream target of METTL3. Furthermore, the m6A-YTHDF2-dependent pathway is downregulated to decrease HMGB1. Targeting HMGB1, combined with gemcitabine and ferroptosis inducer RSL3, can significantly inhibit tumor proliferation. Furthermore, we prove that METTL3 exists in O-GlcNAcylation, and stabilizes its expression, the S118 site O-GlcNAcylation maintains the stability of the METTL3 protein, the specific mechanism lies in that O-GlcNAcylation interferes with ubiquitination, i.e., O-GlcNAcylation enhances the interaction between METTL3 and deubiquitinase EIF3H, resulting in a lower level of ubiquitination modification and enhanced stability of the METTL3 protein (Fig. 6 K). These findings provide new insights into the treatment of pancreatic cancer. In recent years, post-translational modification of METTL3 has attracted more and more attention from academia. SUMOylation of METTL3 at K177, K211, K212 and K215 sites might repress its methyltransferase activity for m6A RNA methylation. Subsequent SUMOylation of METTL3 promotes colony formation and tumor growth of human non-small cell lung cancer (NSCLC) H1299 cells 37 , which is caused by downstream gene dysregulation. Lacylation-driven METTL3-mediated RNA m6A modification plays an important role in promoting the immunosuppressive ability of tumor-infiltrating myeloid cells 38 . In addition, METTL3 can bind to USP5, and this binding is promoted by ERK-mediated phosphorylation. ERK-dependent METTL3 stabilization affects cellular mRNA m6A methylation which could contribute to tumorigenesis 39 . The E3 ubiquitin ligase RNF113A mediates the ubiquitin/proteasome-dependent degradation of METTL3 through the K48-linked multi-ubiquitin chain, reducing the level of m6A modification to promote the inhibition of acute myeloid leukemia 40 . Our study revealed that OGT induced O-GlcNAcylation of METTL3 by interacting with METTL3, increasing the stability of METTL3 protein. In addition, we also found that METTL3 was O-GlcNAcylated at Ser118 site and emphasized its role in crosstalk ubiquitination. The improvement of METTL3 stability led to the enhancement of the level of m6A modification of downstream target genes. These findings also further clarify the regulatory network of post-translational modification of proteins and m6A modification of RNA in the pathogenesis of pancreatic cancer. O-GlcNAcylation has been demonstrated to impact various functional activities of proteins, including stability, transcriptional activity, localization, and protein-protein interactions 17 . The presence of O-GlcNAcylation at Thr58 competes with phosphorylation, leading to enhanced protein stability and hindered degradation by proteasomes for c-MYC 41 . Inhibition of O-GlcNAcase (OGA) activity results in prolonged half-life of YAP through the inhibition of SCF β−TRCP E3-ubiquitin ligase 42 . Moreover, accumulating evidence suggests that elevated levels of O-GlcNAcylation are closely associated with pancreatic cancer progression, metastasis and recurrence, ECM remodeling, and immunotherapy resistance 43,44 .In our study on pancreatic cancer, we observed a regulatory relationship between METTL3 expression and the extent of O-GlcNAcylation modification. Specifically, enhancing O-GlcNAcylation of METTL3 through exogenous transfection of OGT increased its protein stability. Conversely, loss-of-function mutation in the catalytic domain of OGT (K908A) reduced METTL3 expression. Similarly, treatment with PugNAc (an inhibitor targeting OGA) upregulated METTL3 expression. Deficiency in O-GlcNAcylation shortened the half-life of METTL3 by promoting K48-linked ubiquitination. Additionally, loss-of-function mutation S118A resulted in enhanced stability of METTL3 by inhibiting its binding to EIF3H protein via abrogating the interaction with this partner molecule. Further investigations are required to determine whether degradation-induced regulation downstream phenotypes occur due to loss-of-O-GlcNAcylation caused by S118A mutation. Interestingly, the S118A mutant does not completely abolish O-GlcNAcylation of METTL3, thereby implying the existence of potential alternative O-GlcNAc sites. High mobility group box 1 (HMGB1) is a non-histone chromatin-associated protein that is widely distributed in eukaryotic cells and plays a role in DNA damage repair and genomic stability maintenance 45 . HMGB1 appears to have conflicting functions in the progression and treatment of cancer. On the one hand, HMGB1 may promote tumor formation, for example, by playing an important role in regulating mouse oval cell activation and liver cancer development related to inflammation 46 . On the other hand, LPS induces pro-inflammatory cytokines (such as IL-1β, IL-6, and TNF-α) in a HMGB1-dependent manner to improve colorectal cancer progression 47 . In terms of resistance, both the nuclear and cytoplasmic HMGB1 promote autophagy and inhibit tumor cell apoptosis to induce chemotherapy resistance 48 . The role of HMGB1 in ferroptosis is also increasingly reported. Neutrophil extracellular traps mediate cardiomyocyte ferroptosis via the Hippo-Yap pathway to exacerbate doxorubicin-induced cardiotoxicity 49 , where HMGB1 acts as an iron apoptosis inducer by upregulating ferroptosis in astrocytes, thereby aggravating the acute injury after ischemia in the brain 50 . In pancreatic ductal adenocarcinoma, Targeting the MCP-GPX4/HMGB1 Axis for Effectively Triggering Immunogenic Ferroptosis 13 ; N6F11 treatment caused ferroptotic cancer cell death that initiated HMGB1-dependent antitumor immunity mediated by CD8 + T cells 51 . Previous studies have shown that post-translational modifications (i.e., acetylation, phosphorylation, and methylation) of HMGB1 near or within its nuclear localization sequences (NLS) can induce its translocation to the cytoplasm, leading to the subsequent release of HMGB1 during inflammation 52–54 . In sepsis, YTHDF2 inhibits the release of HMGB1 and alleviates inflammatory responses 55 , but no further exploration has been made on the mechanism of YTHDF2 regulating HMGB1 expression; in primary liver cancer, HMGB1 is demethylated by ALKBH5 and recognized by Reader YTHDF2, promoting its degradation 56 . Here, we demonstrate for the first time that HMGB1 is a target gene of METTL3 and is degraded by METTL3 in a m6A-YTHDF2-dependent manner. Furthermore, we found that the supplementation of HMGB1 could alleviate the inhibition of ferroptosis by METTL3 in pancreatic cancer. Our study reveals that HMGB1 is a target protein of Writer METTL3 and proves that METTL3 regulates HMGB1 in a m6A-dependent manner, thereby regulating ferroptosis. This provides a certain basis for a new treatment direction in pancreatic cancer. In summary, we have elucidated the molecular mechanism underlying METTL3-mediated regulation of ferroptosis and gemcitabine resistance in pancreatic cancer. Our findings demonstrate that elevated expression of METTL3 in pancreatic cancer is partially dependent on O-GlcNAcylation, which is mediated by OGT. The stabilization of METTL3 protein expression through O-GlcNAcylation at the S118 site enhances its interaction with deubiquitinase EIF3H. Moreover, we have discovered that METTL3 promotes the degradation of ferroptosis driver HMGB1 in an m6A-YTHDF2-dependent manner. Additionally, our study reveals that a combination therapy targeting HMGB1, along with gemcitabine and the ferroptosis inducer RSL3, effectively suppresses pancreatic cancer development. However, our study has certain limitations including insufficient exploration of downstream mechanisms involved in regulating ferroptosis and gemcitabine resistance upon O-GlcNAcylation-induced increase in METTL3 stability. Furthermore, clinical data supporting these findings are lacking in our dataset. In future studies, we aim to investigate whether O-GlcNAcylation of METTL3 can regulate its resistance to ferroptosis and gemcitabine treatment while also exploring its impact on the tumor microenvironment and potential modulation of immune checkpoint blockade efficacy through an m6A-dependent mechanism. These efforts will provide valuable insights into the potential clinical application of METTL3 for treating pancreatic cancer and improving treatment outcomes. Declarations Ethical and Legal Declarations This study and included experimental procedures were approved by The Second Affiliated Hospital of Kunming Medical University. All animal experiments were approved by the Animal Care and Use Committee of the Ethical Institution of The Second Affiliated Hospital of Kunming Medical University . Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors confirm that there are no conflicts of interest. Funding This project is supported by Yunnan Provincial Applied Basic Research Plan (202401AT070020), Yunnan Provincial Science and Technology Department Basic Research Plan (202101AY070001-142), Kunkun-Medical joint special project number: 202301AY070001-270, 202101AY070001-145, Scientific Research Foundation of Education Department of Yunnan Province (2024J0346), Academician Expert Workstation of Yunnan Province (202205AF150127), Supported by Weichuang Treatment Innovation Team of Hepatobiliary and Pancreatic Surgery Department of Yunnan Province (202405AS350021) Author contributions QiuhongWang, Dongyun Cun, Dong Wei and Xiawei Yang made contribution to the conception and design; Chunman Li, Kun Su, Tao Wang, Renchao Zou and Lianmin Wang analyzed and interpreted data; QiuhongWang drafted the article; Tao Wu, Bo Tang and Dongyun Cun revisied it critically for important intellectual content; All authors approved the final version to be published. Acknowledgements Not applicable. 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Ito, I., Fukazawa, J. & Yoshida, M. Post-translational methylation of high mobility group box 1 (HMGB1) causes its cytoplasmic localization in neutrophils. J Biol Chem 282 , 16336-16344, doi:10.1074/jbc.M608467200 (2007). Kang, H. J. et al. Non-histone nuclear factor HMGB1 is phosphorylated and secreted in colon cancers. Lab Invest 89 , 948-959, doi:10.1038/labinvest.2009.47 (2009). Zeng, Z. et al. The m6A reader YTHDF2 alleviates the inflammatory response by inhibiting IL-6R/JAK2/STAT1 pathway-mediated high-mobility group box-1 release. Burns Trauma 11 , tkad023, doi:10.1093/burnst/tkad023 (2023). Chen, G. et al. ALKBH5-Modified HMGB1-STING Activation Contributes to Radiation Induced Liver Disease via Innate Immune Response. Int J Radiat Oncol Biol Phys 111 , 491-501, doi:10.1016/j.ijrobp.2021.05.115 (2021). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryFigure1.jpg Fig S1 METTL3 is upregulated in pancreatic cancer and promotes pancreatic cancer cell proliferation, stemness, and gemcitabine resistance A: mRNA level of METTL3 in 7 pairs of pancreatic cancer samples. B: Western Blot detection of METTL3 expression. C, D: Cell viability assays using CCK-8 to detect stable overexpression and stable knockdown of METTL3 in (C)PANC1 and (D)BXPC3 cells. E: Quantification of the diamater of cell spheroids in pancreatic cancer cell lines PANC1 and BXPC3 after METTL3 knockout. F: Quantification chart of the percentage of CD133 + pancreatic cancer cells after METTL3 knockout. ***p < 0.001, **p < 0.01, *p 0.05. SupplementaryFigure2.jpg Fig S2 The regulation of ferroptosis is mediated by METTL3. A：KEGG analysis based on the overlapping genes of METTL3 from the top 2000 differential genes ranked in GSE146806 siNC VC siMETTL3 and the genes corresponding to the RNA binding peaks bound by METTL3 in GSE132306. SupplementaryFigure3.jpg Fig S3 The inhibition of ferroptosis in pancreatic cancer cells is mediated by METTL3 A: Representative images and quantification of mitochondrial superoxide levels in BXPC3 cells with stable knockdown or overexpression of METTL3. B: Measurement of MDA levels in BXPC3 cells with stable knockdown or overexpression of METTL3. C: Measurement of GSH levels in BXPC3 cells with stable knockdown or overexpression of METTL3. D: Measurement and quantification of reactive oxygen species (ROS) levels in BXPC3 cells with stable knockdown or overexpression of METTL3. E: Detection and quantification of ferrous ion levels in BXPC3 cells with stable knockdown or overexpression of METTL3 using FerroOrange dye. F: Measurement of mitochondrial membrane potential in BXPC3 cells with stable knockdown or overexpression of METTL3. ***p < 0.001, **p < 0.01, *p 0.05. SupplementaryFigure4.jpg Fig S4 HMGB1 was identified as a downstream target of METTL3 A: Quantitative determination of m6A in BXPC3 cells with stable knockout and overexpression of METTL3. B: RIP-qPCR validation of METTL3-regulated HMGB1 m6A modification in BXPC3 cells. C: The m6A level of HMGB1 mRNA when the expression of METTL3 was altered in PANC1 cells, as detected by MeRIP-qPCR. D: The m6A level of HMGB1 mRNA when the expression of METTL3 was altered in BXPC3 cells, as detected by MeRIP-qPCR. E: Protein levels of HMGB1 after knockout or overexpression of METTL3 in PANC1 and BXPC3 cells. F: mRNA levels of HMGB1 after knockout or overexpression of METTL3 in PANC1 and BXPC3 cells. G: schematic diagram of luciferase reporter with wild type and mutated 3’UTR of HMGB1. The wild-type or mutant versions of HMGB1 3’UTR were cloned into a plasmid. For mutant versions, 2 putative N6-methyladenosine-modified adenosines were mutated to cytosines H: The m6A level and METTL3 binding level of HMGB1 mRNA when the m6A methylation sites on HMGB1 mRNA were mutated in BXPC3 cells detected by MeRIP-qPCR. I: Mutation of m6A methylation sites on HMGB1 mRNA contributes to the enhancement of HMGB1 mRNA stability in BXPC3 cells. ***p < 0.001, **p < 0.01, *p 0.05. SupplementaryFigure5.jpg Fig S5 Inhibition of ferroptosis by METTL3 and gemcitabine resistance are mitigated by HMGB1 A: Docking model of HMGB1 and gemcitabine. B: Quantification chart of CD133+ percentage in PANC1 and BXPC3 cells with stable overexpression of METTL3, transfected with or without HMGB1. C: Quantification chart of tumor volume (n=4). D: Quantification chart of tumor weight (n=4). ***p < 0.001, **p < 0.01, *p 0.05. SupplementaryFigure6.jpg FigS6 OGT was upregulated in pancreatic cancer A: The UALCAN database (https://ualcan.path.uab.edu/index.html) shows that OGT protein is upregulated in pancreatic cancer. SupplementaryFigure7.jpg Fig S7 O-GlcNAcylation stabilizes METTL3 protein via enhancing the interaction between METTL3 and EIF3H A: Docking model of METTL3 and OGT interaction. B: Interaction area and binding free energy of the relevant complex. C: Truncated variants of OGT. D: Co-IP assay performed in HEK293T cells transfected with Flag-METTL3 and truncated HA-OGT to detect protein interactions. E: Correlation analysis of METTL3 and OGT protein levels in pancreatic cancer. F: Quantification of METTL3 expression levels under corresponding treatments through qPCR experiments. G: PANC1 cells transfected with MYC-OGT, HA-UB, HA-UB K48, and HA-UB K63 were subjected to protein A/G agarose immunoprecipitation using anti-METTL3 antibodies, followed by HA western blotting. H: Venn diagram showing METTL3 mass spectrometry results (PXD036899). METTL3 interacting proteins were obtained from the database https://thebiogrid.org/, and all DUBs were retrieved from the database http://iuucd.biocuckoo.org/. Overlapping gene EIF3H was identified. I: Correlation analysis of EIF3H and METTL3 expression obtained from GEPIA2 (http://gepia2.cancer-pku.cn/#index). J: METTL3 and EIF3H protein expression in pancreatic cancer obtained from the Proteomic Data Commons database (cancer.gov), with correlation analysis plotted. K: METTL3-WT or METTL3-S118A mutants were transfected into PANC1 and BXPC3 cells. Flag immunoprecipitation was performed, followed by western blotting with the corresponding antibodies. L: PANC1 cells were transfected with METTL3-WT or METTL3-S118A plasmids. METTL3 ubiquitination was detected by immunoprecipitation using anti-Flag antibodies. ***p < 0.001, **p < 0.01, *p 0.05. supplementarytable1.docx Supplementary table1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5606582","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":390211993,"identity":"0045c9a3-19d0-45d1-949f-0c7633df9d6c","order_by":0,"name":"qiuhong 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Tang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Tang","suffix":""},{"id":390212003,"identity":"89001dda-5d97-4095-98af-e43227f65560","order_by":10,"name":"Tao Wu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-12-09 07:10:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5606582/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5606582/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71596034,"identity":"5686818b-a14f-40e5-9154-5985c2f00590","added_by":"auto","created_at":"2024-12-17 04:22:13","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2795327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMETTL3 is upregulated in pancreatic cancer and promotes pancreatic cancer cell proliferation, stemness, and gemcitabine resistance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: METTL3 related survival curves of pancreatic cancer patients in the PanCanSurvPlot database.\u003c/p\u003e\n\u003cp\u003eB: Immunohistochemical analysis of METTL3 expression in pancreatic cancer tissues.\u003c/p\u003e\n\u003cp\u003eC: Western blot detection and quantification of METTL3 expression in 4 pairs of pancreatic cancer tissues.\u003c/p\u003e\n\u003cp\u003eD: EDU assay of pancreatic cancer cell lines PANC1 and BXPC3 after METTL3 knockout.\u003c/p\u003e\n\u003cp\u003eE: Sensitivity of PANC1 cells with stable knockout of METTL3, stable overexpression of METTL3, and their corresponding controls to gemcitabine.\u003c/p\u003e\n\u003cp\u003eF: Sensitivity of BXPC3 cells with stable knockout of METTL3, stable overexpression of METTL3, and their corresponding controls to gemcitabine\u003c/p\u003e\n\u003cp\u003eG: Cell spheroid assay of pancreatic cancer cell lines PANC1 and BXPC3 after METTL3 knockout.\u003c/p\u003e\n\u003cp\u003eH: Percentage of CD133+ pancreatic cancer cells after METTL3 knockout.\u003c/p\u003e\n\u003cp\u003e***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05, ns p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/87575164a5d654ea6e82bc5b.jpg"},{"id":71595899,"identity":"a1076c6f-f57c-42a1-8e3c-d7a4e33cd1b7","added_by":"auto","created_at":"2024-12-17 04:14:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1701010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe inhibition of ferroptosis in pancreatic cancer cells is mediated by METTL3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Representative images and quantification of mitochondrial superoxide levels in PANC1 cells with stable knockdown or overexpression of METTL3.\u003c/p\u003e\n\u003cp\u003eB: Measurement of MDA levels in PANC1 cells with stable knockdown or overexpression of METTL3.\u003c/p\u003e\n\u003cp\u003eC: Measurement of GSH levels in PANC1 cells with stable knockdown or overexpression of METTL3.\u003c/p\u003e\n\u003cp\u003eD: Measurement and quantification of reactive oxygen species (ROS) levels in PANC1 cells with stable knockdown or overexpression of METTL3.\u003c/p\u003e\n\u003cp\u003eE: Detection and quantification of ferrous ion levels in PANC1 cells with stable knockdown or overexpression of METTL3 using FerroOrange dye.\u003c/p\u003e\n\u003cp\u003eF: Measurement of mitochondrial membrane potential in PANC1 cells with stable knockdown or overexpression of METTL3. ***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05, ns p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/6ade0921b4a12c34463e8cc9.jpg"},{"id":71595893,"identity":"3229378a-7e55-4e00-ab2e-257519d165d9","added_by":"auto","created_at":"2024-12-17 04:14:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1159615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHMGB1 was identified as a downstream target of METTL3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Quantitative determination of m6A in PANC1 cells with stable knockout and overexpression of METTL3.\u003c/p\u003e\n\u003cp\u003eB: Venn diagram showing differential genes obtained from GSE146806 analysis, correlation genes of METTL3 in pancreatic cancer obtained from UALCAN(https://ualcan.path.uab.edu/index.html), target genes from RM2Target database, and the genes corresponding to METTL3 binding RNA peaks in GSE13306.\u003c/p\u003e\n\u003cp\u003eC: RIP-qPCR validation of METTL3-regulated HMGB1 m6A modification in PANC1 cells.\u003c/p\u003e\n\u003cp\u003eD: RNA pull-down experiments demonstrate the interaction between METTL3 and HMGB1 mRNA in PANC1 and BXPC3 cells.\u003c/p\u003e\n\u003cp\u003eE: Deletion of METTL3 enhances the stability of HMGB1 mRNA in PANC1 cells.\u003c/p\u003e\n\u003cp\u003eF: Deletion of METTL3 enhances the stability of HMGB1 mRNA in BXPC3 cells.\u003c/p\u003e\n\u003cp\u003eG: Relative luciferase activity of HMGB1 3’UTR with wild-type or mutated m6A sites after METTL3 silencing in PANC1 cells.\u003c/p\u003e\n\u003cp\u003eH: The m6A level and METTL3 binding level of HMGB1 mRNA when the m6A methylation sites on HMGB1 mRNA were mutated in PANC1 cells detected by MeRIP-qPCR.\u003c/p\u003e\n\u003cp\u003eI: Mutation of m6A methylation sites on HMGB1 mRNA contributes to the enhancement of HMGB1 mRNA stability in PANC1 cells.\u003c/p\u003e\n\u003cp\u003e***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05, ns p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/abec304ba76cb698262d5a24.jpg"},{"id":71595897,"identity":"a43a7ba9-0b59-46de-a2c0-4026f5958f7f","added_by":"auto","created_at":"2024-12-17 04:14:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1198948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYTHDF2 mediates HMGB1 mRNA expression in an m6A-dependent manner\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: RNA pull-down experiments demonstrate the interaction between YTHDF2 and HMGB1 mRNA in PANC1 cells and BXPC3 cells.\u003c/p\u003e\n\u003cp\u003eB: mRNA levels of HMGB1 after interference with YTHDF2 in PANC1 and BXPC3 cells.\u003c/p\u003e\n\u003cp\u003eC, D: Protein levels of HMGB1 after interference with YTHDF2 in PANC1 and BXPC3 cells.\u003c/p\u003e\n\u003cp\u003eE: The correlation between YTHDF2 and HMGB1 protein expression in pancreatic cancer.\u003c/p\u003e\n\u003cp\u003eF: RIP-qPCR validation of YTHDF1-regulated HMGB1 m6A modification in PANC1 and BXPC3 cells.\u003c/p\u003e\n\u003cp\u003eG: Relative luciferase activity of HMGB1 3’UTR with wild-type or mutated m6A sites after YTHDF2 silencing in PANC1 and BXPC3 cells.\u003c/p\u003e\n\u003cp\u003eH: Interference with YTHDF2 increases the stability of HMGB1 mRNA in PANC1 and BXPC3 cells.\u003c/p\u003e\n\u003cp\u003e***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05, ns p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/f9a815d83b9a4d2de5e78a48.jpg"},{"id":71595901,"identity":"6d77ed05-83f5-4533-b1d4-dadfac807633","added_by":"auto","created_at":"2024-12-17 04:14:13","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3311725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of ferroptosis by METTL3 and gemcitabine resistance are mitigated by HMGB1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Sensitivity to gemcitabine in PANC1 cells with stable overexpression of METTL3 and corresponding controls, with or without transfection of HMGB1.\u003c/p\u003e\n\u003cp\u003eB: Sensitivity to gemcitabine in BXPC3 cells with stable overexpression of METTL3 and corresponding controls, with or without transfection of HMGB1.\u003c/p\u003e\n\u003cp\u003eC: Quantification of cell spheroid diameter in PANC1 and BXPC3 cells with stable overexpression of METTL3, transfected with or without HMGB1.\u003c/p\u003e\n\u003cp\u003eD: Quantification of ROS levels in PANC1 and BXPC3 cells with stable overexpression of METTL3, transfected with or without HMGB1.\u003c/p\u003e\n\u003cp\u003eE: Quantification of MDA and GSH levels in PANC1 and BXPC3 cells with stable overexpression of METTL3, transfected with or without HMGB1.\u003c/p\u003e\n\u003cp\u003eF: Effect on cell viability of PANC1 cells with stable overexpression of METTL3 and corresponding controls, with or without transfection of HMGB1, treated with increasing concentrations of gemcitabine, with or without RSL3 treatment.\u003c/p\u003e\n\u003cp\u003eG: Effect on cell viability of BXPC3 cells with stable overexpression of METTL3 and corresponding controls, with or without transfection of HMGB1, treated with increasing concentrations of gemcitabine, with or without RSL3 treatment.\u003c/p\u003e\n\u003cp\u003eH: Representative images of the tumors after respective treatments.\u003c/p\u003e\n\u003cp\u003eI: Representative images of ki67 and TUNEL.\u003c/p\u003e\n\u003cp\u003e***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05, ns p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/4edd6c4dee15f20e066ae311.jpg"},{"id":71595902,"identity":"d8f755e5-89e7-489d-81a2-8a36270184a8","added_by":"auto","created_at":"2024-12-17 04:14:13","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1708712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eO-GlcNAcylation stabilizes METTL3 protein via enhancing the interaction between METTL3 and EIF3H\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Co-IP assay was performed in HEK293T cells transfected with HA-OGT, HA-OGT-K908A, or Flag-METTL3 plasmids to detect protein interactions.\u003c/p\u003e\n\u003cp\u003eB: siNC, siOGT, or the K908A mutant were transfected into PANC1 cells, followed by treatment with or without the OGA inhibitor PugNAc (1µM, 4 h). Cell lysates were then subjected to immunoblotting using the indicated antibodies.\u003c/p\u003e\n\u003cp\u003eC: Western blot analysis was used to detect the expression level of METTL3 protein under corresponding treatments.\u003c/p\u003e\n\u003cp\u003eD: siNC, siOGT, or the K908A mutant were transfected into PANC1 cells and treated with CHX (300µg/ml) for designated time periods to assess METTL3 protein levels.\u003c/p\u003e\n\u003cp\u003eE: PANC1 cells treated with siNC/siOGT or K908A and HA-ub were immunoprecipitated with protein A/G agarose and incubated with anti-METTL3 antibodies, followed by HA western blotting.\u003c/p\u003e\n\u003cp\u003eF: Transfect PANC1 cells with siEIF3H or siNC, and detect METTL3 ubiquitination by immunoprecipitation with anti-Flag antibody.\u003c/p\u003e\n\u003cp\u003eG: PANC1 cells transfected with HA-OGT were immunoprecipitated with protein A/G agarose and incubated with anti-METTL3 antibodies. Western blotting for METTL3 and EIF3H was then performed.\u003c/p\u003e\n\u003cp\u003eH: PANC1 cells treated with PugNAc (1µM) were immunoprecipitated with protein A/G agarose and incubated with anti-METTL3 antibodies. Western blotting for METTL3 and EIF3H was subsequently conducted.\u003c/p\u003e\n\u003cp\u003eI: METTL3-WT or METTL3-S118A mutants were transfected into PANC1 cells, and treated with CHX (300µg/ml) for designated time periods to monitor METTL3 protein levels.\u003c/p\u003e\n\u003cp\u003eJ: METTL3-WT or METTL3-S118A mutants were transfected into PANC1 cells. Protein A/G agarose immunoprecipitation was performed with anti-METTL3 antibodies, followed by EIF3H western blotting.\u003c/p\u003e\n\u003cp\u003eK: Schematic diagram illustrating the mechanism.\u003c/p\u003e\n\u003cp\u003e***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05, ns p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/19631363c26cab0dc2314d51.jpg"},{"id":71597340,"identity":"045ce171-c815-4bd6-ae5a-db5a66d60bf7","added_by":"auto","created_at":"2024-12-17 04:38:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12921369,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/4ae44655-22b5-4ccf-95fa-3b4ce02cc919.pdf"},{"id":71596032,"identity":"cd868f70-8d08-472c-9bb8-e5544c9b0476","added_by":"auto","created_at":"2024-12-17 04:22:13","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":872581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig S1 METTL3 is upregulated in pancreatic cancer and promotes pancreatic cancer cell proliferation, stemness, and gemcitabine resistance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: mRNA level of METTL3 in 7 pairs of pancreatic cancer samples.\u003c/p\u003e\n\u003cp\u003eB: Western Blot detection of METTL3 expression.\u003c/p\u003e\n\u003cp\u003eC, D: Cell viability assays using CCK-8 to detect stable overexpression and stable knockdown of METTL3 in (C)PANC1 and (D)BXPC3 cells.\u003c/p\u003e\n\u003cp\u003eE: Quantification of the diamater of cell spheroids in pancreatic cancer cell lines PANC1 and BXPC3 after METTL3 knockout.\u003c/p\u003e\n\u003cp\u003eF: Quantification chart of the percentage of CD133\u003csup\u003e+\u003c/sup\u003e pancreatic cancer cells after METTL3 knockout.\u003c/p\u003e\n\u003cp\u003e***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05, ns p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/1a8301da2256cc1afe92f400.jpg"},{"id":71596033,"identity":"96416ef6-220e-497d-b062-484959dd7b9a","added_by":"auto","created_at":"2024-12-17 04:22:13","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":563014,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig S2 The regulation of ferroptosis is mediated by METTL3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA：KEGG analysis based on the overlapping genes of METTL3 from the top 2000 differential genes ranked in GSE146806 siNC VC siMETTL3 and the genes corresponding to the RNA binding peaks bound by METTL3 in GSE132306.\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/e8ef0b2bc5168626434f9382.jpg"},{"id":71595892,"identity":"700e2f49-6c7b-4aaf-b7a0-a59f0ed5f6c6","added_by":"auto","created_at":"2024-12-17 04:14:13","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1836575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig S3 The inhibition of ferroptosis in pancreatic cancer cells is mediated by METTL3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Representative images and quantification of mitochondrial superoxide levels in BXPC3 cells with stable knockdown or overexpression of METTL3.\u003c/p\u003e\n\u003cp\u003eB: Measurement of MDA levels in BXPC3 cells with stable knockdown or overexpression of METTL3.\u003c/p\u003e\n\u003cp\u003eC: Measurement of GSH levels in BXPC3 cells with stable knockdown or overexpression of METTL3.\u003c/p\u003e\n\u003cp\u003eD: Measurement and quantification of reactive oxygen species (ROS) levels in BXPC3 cells with stable knockdown or overexpression of METTL3.\u003c/p\u003e\n\u003cp\u003eE: Detection and quantification of ferrous ion levels in BXPC3 cells with stable knockdown or overexpression of METTL3 using FerroOrange dye.\u003c/p\u003e\n\u003cp\u003eF: Measurement of mitochondrial membrane potential in BXPC3 cells with stable knockdown or overexpression of METTL3.\u003c/p\u003e\n\u003cp\u003e***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05, ns p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"SupplementaryFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/f56acb72889e151b3aa306a9.jpg"},{"id":71595906,"identity":"0714c27c-16c8-46f4-a372-65129833f17e","added_by":"auto","created_at":"2024-12-17 04:14:13","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1165123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig S4 HMGB1 was identified as a downstream target of METTL3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Quantitative determination of m6A in BXPC3 cells with stable knockout and overexpression of METTL3.\u003c/p\u003e\n\u003cp\u003eB: RIP-qPCR validation of METTL3-regulated HMGB1 m6A modification in BXPC3 cells.\u003c/p\u003e\n\u003cp\u003eC: The m6A level of HMGB1 mRNA when the expression of METTL3 was altered in PANC1 cells, as detected by MeRIP-qPCR.\u003c/p\u003e\n\u003cp\u003eD: The m6A level of HMGB1 mRNA when the expression of METTL3 was altered in BXPC3 cells, as detected by MeRIP-qPCR.\u003c/p\u003e\n\u003cp\u003eE: Protein levels of HMGB1 after knockout or overexpression of METTL3 in PANC1 and BXPC3 cells.\u003c/p\u003e\n\u003cp\u003eF: mRNA levels of HMGB1 after knockout or overexpression of METTL3 in PANC1 and BXPC3 cells.\u003c/p\u003e\n\u003cp\u003eG: schematic diagram of luciferase reporter with wild type and mutated 3’UTR of HMGB1. The wild-type or mutant versions of HMGB1 3’UTR were cloned into a plasmid. For mutant versions, 2 putative N6-methyladenosine-modified adenosines were mutated to cytosines\u003c/p\u003e\n\u003cp\u003eH: The m6A level and METTL3 binding level of HMGB1 mRNA when the m6A methylation sites on HMGB1 mRNA were mutated in BXPC3 cells detected by MeRIP-qPCR.\u003c/p\u003e\n\u003cp\u003eI: Mutation of m6A methylation sites on HMGB1 mRNA contributes to the enhancement of HMGB1 mRNA stability in BXPC3 cells.\u003c/p\u003e\n\u003cp\u003e***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05, ns p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"SupplementaryFigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/9fc07d4d34e9385048bd8177.jpg"},{"id":71596035,"identity":"8d1adb59-4943-4043-91fd-8b0dbcdb9ec4","added_by":"auto","created_at":"2024-12-17 04:22:13","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1044307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig S5 Inhibition of ferroptosis by METTL3 and gemcitabine resistance are mitigated by HMGB1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Docking model of HMGB1 and gemcitabine.\u003c/p\u003e\n\u003cp\u003eB: Quantification chart of CD133+ percentage in PANC1 and BXPC3 cells with stable overexpression of METTL3, transfected with or without HMGB1.\u003c/p\u003e\n\u003cp\u003eC: Quantification chart of tumor volume (n=4).\u003c/p\u003e\n\u003cp\u003eD: Quantification chart of tumor weight (n=4).\u003c/p\u003e\n\u003cp\u003e***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05, ns p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"SupplementaryFigure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/6a83df8ad0e5487a0cc1c7ac.jpg"},{"id":71595903,"identity":"c33a373e-19a6-45e2-960e-5c6bfd7a24a3","added_by":"auto","created_at":"2024-12-17 04:14:13","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":257146,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigS6 OGT was upregulated in pancreatic cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: The UALCAN database (https://ualcan.path.uab.edu/index.html) shows that OGT protein is upregulated in pancreatic cancer.\u003c/p\u003e","description":"","filename":"SupplementaryFigure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/993ee60bca1fe1c2de6d5b56.jpg"},{"id":71595900,"identity":"292b1555-e09f-4e67-8d88-fa30a7167bc9","added_by":"auto","created_at":"2024-12-17 04:14:13","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1913317,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig S7 O-GlcNAcylation stabilizes METTL3 protein via enhancing the interaction between METTL3 and EIF3H\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Docking model of METTL3 and OGT interaction.\u003c/p\u003e\n\u003cp\u003eB: Interaction area and binding free energy of the relevant complex.\u003c/p\u003e\n\u003cp\u003eC: Truncated variants of OGT.\u003c/p\u003e\n\u003cp\u003eD: Co-IP assay performed in HEK293T cells transfected with Flag-METTL3 and truncated HA-OGT to detect protein interactions.\u003c/p\u003e\n\u003cp\u003eE: Correlation analysis of METTL3 and OGT protein levels in pancreatic cancer.\u003c/p\u003e\n\u003cp\u003eF: Quantification of METTL3 expression levels under corresponding treatments through qPCR experiments.\u003c/p\u003e\n\u003cp\u003eG: PANC1 cells transfected with MYC-OGT, HA-UB, HA-UB K48, and HA-UB K63 were subjected to protein A/G agarose immunoprecipitation using anti-METTL3 antibodies, followed by HA western blotting.\u003c/p\u003e\n\u003cp\u003eH: Venn diagram showing METTL3 mass spectrometry results (PXD036899). METTL3 interacting proteins were obtained from the database https://thebiogrid.org/, and all DUBs were retrieved from the database http://iuucd.biocuckoo.org/. Overlapping gene EIF3H was identified.\u003c/p\u003e\n\u003cp\u003eI: Correlation analysis of EIF3H and METTL3 expression obtained from GEPIA2 (http://gepia2.cancer-pku.cn/#index).\u003c/p\u003e\n\u003cp\u003eJ: METTL3 and EIF3H protein expression in pancreatic cancer obtained from the Proteomic Data Commons database (cancer.gov), with correlation analysis plotted.\u003c/p\u003e\n\u003cp\u003eK: METTL3-WT or METTL3-S118A mutants were transfected into PANC1 and BXPC3 cells. Flag immunoprecipitation was performed, followed by western blotting with the corresponding antibodies.\u003c/p\u003e\n\u003cp\u003eL: PANC1 cells were transfected with METTL3-WT or METTL3-S118A plasmids. METTL3 ubiquitination was detected by immunoprecipitation using anti-Flag antibodies.\u003c/p\u003e\n\u003cp\u003e***p \u0026lt; 0.001, **p \u0026lt; 0.01, *p \u0026lt; 0.05, ns p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"SupplementaryFigure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/b8fb2b758ed43a8a891fb309.jpg"},{"id":71596037,"identity":"bb9e8774-4c7f-436e-be32-fd91b754171a","added_by":"auto","created_at":"2024-12-17 04:22:14","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15575,"visible":true,"origin":"","legend":"Supplementary table1","description":"","filename":"supplementarytable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5606582/v1/33b518dd999fc07ba992737a.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"O-GlcNAcylation with ubiquitination stabilizes METTL3, promoting HMGB1 degradation to inhibit ferroptosis and enhance gemcitabine resistance in pancreatic cancer","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePancreatic cancer is a malignant tumor related to the gastrointestinal system \u003csup\u003e1\u003c/sup\u003e, typically presenting at an advanced metastatic stage, rendering it unresectable and exhibiting limited response to chemotherapy \u003csup\u003e2\u003c/sup\u003e. Surgical resection is not beneficial for over 80% of patients \u003csup\u003e3\u003c/sup\u003e, as those who undergo radical resection face a high risk of recurrence \u003csup\u003e4\u003c/sup\u003e. In recent years, there has been a global increase in the prevalence and mortality rates of pancreatic cancer, with only a 12.8% 5-year survival rate (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://seer.cancer.gov/\u003c/span\u003e\u003cspan address=\"https://seer.cancer.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) observed. Gemcitabine (GEM)-based combined chemotherapy strategies remain the preferred treatment for advanced pancreatic cancer \u003csup\u003e5\u003c/sup\u003e. However, both GEM monotherapy and GEM combined with chemotherapy have shown very limited efficacy in improving the survival rate of patients with advanced PDAC due to GEM resistance \u003csup\u003e6\u003c/sup\u003e. Therefore, overcoming GEM resistance and identifying new targeted molecules against GEM are key challenges in the treatment of pancreatic cancer.\u003c/p\u003e \u003cp\u003eFerroptosis is an iron-dependent form of cell death characterized by excessive lipid peroxidation \u003csup\u003e7\u003c/sup\u003e. It primarily manifests as mitochondrial abnormalities, iron accumulation, and lipid peroxidation leading to plasma membrane rupture\u003csup\u003e8\u003c/sup\u003e. Recent studies have demonstrated the association between ferroptosis and GEM chemotherapy resistance in various tumor types, including pancreatic cancer \u003csup\u003e5\u003c/sup\u003e. Specific investigations have revealed that FBW7 enhances the cytotoxic effect of gemcitabine by activating both ferroptosis and apoptosis\u003csup\u003e9\u003c/sup\u003e, while SLC38A5 regulates ferroptosis to overcome gemcitabine resistance in pancreatic cancer\u003csup\u003e10\u003c/sup\u003e.In recent years, numerous studies have highlighted the role of HMGB1 in regulating cell ferroptosis \u003csup\u003e11,12\u003c/sup\u003e. In pancreatic cancer specifically, there are reports suggesting that targeting the MCP-GPX4/HMGB1 axis effectively triggers immunogenic ferroptosis in pancreatic ductal adenocarcinoma \u003csup\u003e13\u003c/sup\u003e. However, whether HMGB1 can be targeted to inhibit GEM resistance and promote ferroptosis for inhibiting the development of pancreatic cancer remains unexplored. We observed that the binding free energy between HMGB1 and GEM in the protein-molecule docking model is less than zero, indicating a potential interaction between HMGB1 and GEM as a target molecule. This suggests that targeting HMGB1 may promote ferroptosis in pancreatic cancer cells while inhibiting GEM resistance and preventing further progression of this disease.\u003c/p\u003e \u003cp\u003eO-GlcNAcylation is a reversible posttranslational modification of proteins\u003csup\u003e14\u003c/sup\u003e, resulting from glucose metabolism via the hexosamine biosynthesis pathway (HBP), which integrates glucose, amino acids, fatty acids, and nucleotides\u003csup\u003e15\u003c/sup\u003e. The regulation of O-GlcNAcylation involves two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA)\u003csup\u003e16\u003c/sup\u003e. Since its discovery, O-GlcNAcylation has been implicated in various cellular functions including signal transduction, protein localization and stability, transcriptional control, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation of the O-GlcNAc cycle is associated with the progression of diverse diseases such as diabetes mellitus and its complications as well as cancer and cardiovascular/neurodegenerative disorders\u003csup\u003e17\u003c/sup\u003e. In pancreatic cancer specifically, emerging evidence suggests that alterations in O-GlcNAcylation impact disease progression through modulation of different substrates. For instance: promotion of pancreatic tumor growth by regulating malate dehydrogenase 1 through O-GlcNAcylation has been reported\u003csup\u003e18\u003c/sup\u003e; facilitation of ferroptosis in mesenchymal pancreatic cancer cells via ZEB1's O-GlcNAcylation has also been observed\u003csup\u003e19\u003c/sup\u003e; additionally,O-GlcNAcylation-mediated stabilization of SIRT7 promotes pancreatic cancer progression by disrupting the SIRT7-REGγ interaction\u003csup\u003e20\u003c/sup\u003e. Therefore,it is imperative to identify novel substrate proteins for targeted regulation of pancreatic cancer progression.\u003c/p\u003e \u003cp\u003eM6A is the most prevalent epigenetic modification in eukaryotic cells and governs diverse biological processes \u003csup\u003e21\u003c/sup\u003e. The three primary regulatory factors of M6A epigenetic alterations are the M6A methyltransferase (writer), M6A demethylase (eraser), and M6A recognition protein (reader) \u003csup\u003e22,23\u003c/sup\u003e. METTL3 functions as an S-adenosylmethionine (SAM) binding protein and acts as a catalyst by utilizing its internal SAM-binding domain to transfer methyl from SAM to the adenine base of RNA, resulting in the production of S-adenosylhomocysteine (SAH)\u003csup\u003e21\u003c/sup\u003e. Emerging evidence suggests that METTL3 plays a crucial role in cancer development, acting either as an oncogene or tumor suppressor gene\u003csup\u003e24\u003c/sup\u003e, for instance: METTL3-mediated m6A modification of HDGF mRNA promotes gastric cancer progression\u003csup\u003e25\u003c/sup\u003e. Wang et al. also reported overexpression of METTL3 in breast cancer and identified BCL2 as a target of METTL3. They demonstrated that elevated m6A modification in Bcl-2 mRNA promoted its translation and ultimately facilitated cancer cell proliferation\u003csup\u003e26\u003c/sup\u003e. METTL3 facilitates m6A modification of suppressor of cytokine signaling 2 (SOCS2) and promotes its mRNA degradation through a YTHDF2-dependent pathway, thereby regulating the progression of liver cancer\u003csup\u003e27\u003c/sup\u003e. Studies have demonstrated that elevated levels of METTL3 expression are correlated with advanced pathological stages in PDAC\u003csup\u003e28\u003c/sup\u003e, as well as resistance to chemotherapy and radiotherapy in pancreatic cancer cells \u003csup\u003e29\u003c/sup\u003e. However, the underlying mechanisms remain elusive. Therefore, comprehending the molecular mechanisms and downstream targets governed by METTL3 in pancreatic cancer regulation may offer novel avenues for the treatment and diagnosis of this disease.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the molecular mechanism underlying the role of METTL3, an M6A WRITER, in pancreatic cancer development and its impact on gemcitabine sensitivity. Our findings demonstrate that METTL3 interacts with OGT and undergoes O-GlcNAcylation, thereby elucidating the specific modification site involved in O-GlcNAcylation of METTL3. This post-translational modification stabilizes METTL3 expression by enhancing its interaction with EIF3H, a deubiquitinating enzyme. Additionally, we discovered that METTL3 promotes HMGB1 degradation through a m6A-YTHDF2-dependent pathway, inhibits ferroptosis in pancreatic cancer cells, and confers resistance to gemcitabine treatment. Overall, our study uncovers a novel regulatory axis involving OGT-METTL3-HMGB1 that governs pancreatic cancer development and highlights the potential therapeutic strategy targeting ferroptosis and gemcitabine.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eCell lines\u003c/strong\u003e\u003cstrong\u003e，\u003c/strong\u003e\u003cstrong\u003ecell cultures and transfer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PANC1 and BXPC3 cell lines, along with the HEK293T cell line, were acquired from a Chinese cell bank situated in Shanghai. These cellular cultures were maintained using Dulbecco\u0026lsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), along with penicillin at a concentration of 100 U/ml and streptomycin at a concentration of 100 \u0026mu;g/ml. The incubation process involved keeping the cells at a temperature of 37℃ within an environment saturated with humidity and containing approximately 5% CO\u003csub\u003e2\u003c/sub\u003e. In order to achieve transient expression of plasmids within HEK293T cells, we employed PEI transfection solution based on findings reported by Zhou et al\u003csup\u003e30\u003c/sup\u003e. When introducing plasmids into various cancerous cell lines, Lipofectamine\u0026trade;️3000 manufactured by Invitrogen was utilized according to guidelines provided by its manufacturer. The siRNA sequences ：siYTHDF2：5\u0026prime;-GCACAGAAGTTGCAAGCAA -3\u0026prime;; siOGT：5\u0026prime;-GCCUGAUAGAUCUGGCAAUTT-3\u0026prime;; \u003c/p\u003e\n\u003cp\u003esiEIF3H: 5ʹ-GCAACTCTTGGAAGAAATATA-3ʹ.\u003c/p\u003e\n\u003cp\u003ePugNAc (ab144670) was purchased from Abcam； Cycloheximide (S7418) was purchased from Selleck. Actinomycin D(50-76-0) was purchased from MCE. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tumor tissue and matched non-tumor tissue samples of pancreatic cancer were obtained from 7 patients who underwent surgery at The Second Affiliated Hospital of Kunming Medical University. All patients provided informed consent and did not receive any preoperative chemotherapy or radiotherapy. This study was approved by the The Second Affiliated Hospital of Kunming Medical University Ethics Review Board. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Viability Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Cell Counting Kit-8 (CCK-8) (B34302, Bimake) was used according to the manufacturer\u0026apos;s instructions. Briefly, cells were seeded in 96-well plates at a density of 1 \u0026times; 104 cells per well. For the gemcitabine treatment group, cells were treated with various concentrations of gemcitabine for a specified duration of 24 hours. Subsequently, CCK-8 reagent (10 \u0026mu;l) was added to each well and incubated in a 5% CO2 incubator at 37\u0026deg;C for 2 hours. The absorbance at 450 nm was measured using a microplate reader. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real time-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cells using TRIzol reagent (Invitrogen, USA). The cDNA synthesis was performed by reverse transcribing the RNA with a reverse transcription kit (Bio-Bio Engineering (Dalian) Co., Ltd.). Real-time fluorescent quantitative PCR was conducted using the SYBR-Green PCR Master Mix kit (TOYOBO, Japan). Please refer to supplementary table 1 for the sequences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were harvested and disrupted using a solution containing SDS. The resulting proteins were separated by electrophoresis on an SDS-PAGE gel and subsequently transferred to a PVDF membrane. The PVDF membrane was then blocked with non-fat milk (5%). Specific primary antibodies were applied to the PVDF membrane for incubation, followed by secondary antibody treatment. Detection of proteins was achieved through chemiluminescence analysis. The following is the antibody information: anti-Flag (F1804) antibodies were purchased from Sigma (MO, USA); anti-\u0026beta;-actin (#4970), anti-O-GlcNAc (#9875) and anti-HMGB1(#3935)were obtained from Cell Signaling Technology (MA, USA), anti-eIF3h (ab60942) was obtained from Abcam (Cambridge, UK). anti-HA (sc-57592) antibodies were purchased from Santa Cruz biotechnology (TX, USA); anti-Myc (60003-2-Ig), anti- METTL3 （15073-1-AP）and anti-YTHDF2(24744-1-AP) were purchased from Proteintech. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCycloheximide treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe corresponding cells were seeded onto 6-well plates and subjected to treatment with cycloheximide (20 mg/mL) for 0, 4, 8, and 12 hours prior to collection. Total proteins were extracted and subsequently analyzed by Western blotting using the respective antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emRNA stability analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActinomycin D, a transcriptional inhibitor commonly employed for RNA stability detection, was administered to transfected cells at a concentration of 5 \u0026mu;g/ml. Subsequently, the cells were collected at designated time intervals and total RNA was extracted using TRIzol reagent. The relative expression levels of HMGB1 mRNA were assessed via qRT-PCR analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eME-ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe EpiQuik\u003csup\u003eTM\u003c/sup\u003e m6A RNA Methylation Quantification Kit (Colorimetric) (Epigentek, USA) is utilized for the colorimetric determination of total m6A levels in pancreatic cancer cells. Specifically, 200 ng of RNA is immobilized onto capture antibodies in each well for subsequent detection. Following multiple incubation steps, the m6A content is quantified using a colorimetric method at 450 nm and calculated based on a standard curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMERIP-qPCR \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MERIP-qPCR procedure was conducted following the guidelines provided by the EpiQuik \u0026trade; CUT\u0026amp;RUN m6A RNA Enrichment (MeRIP) Kit. In brief, an immunocapture solution was prepared by combining reagents in 0.2 ml PCR tubes and rotating them at room temperature for 90 minutes. Each tube received 10 \u0026mu;l of NDE (Nuclear Digestion Enhancer) and 2 \u0026mu;l of CEM (Cleavage Enzyme Mix), followed by a 4-minute incubation at room temperature. The tubes were then placed on a magnetic device until the solution became clear, which took approximately 2 minutes. After discarding the supernatant, samples underwent three washes with 150 \u0026mu;l of WB (Wash Buffer), followed by one wash with 150 \u0026mu;l of PDB (Protein Digestion Buffer). Subsequently, samples were mixed with 20 \u0026mu;l of Protein Digestion Solution and incubated at a temperature of 55 ◦C for a duration of 15 minutes using a thermocycler without a heated lid. RPS (RNA Purification Solution) and absolute ethanol were applied to the samples to resuspend and cleanse the RNA Binding Beads through vortexing. The resuspended beads were then subjected to treatment with Elution Buffer totaling13 \u0026mu;l, allowing for an incubation period at room temperature lasting for5 minutes to release RNA from the beads.Finally,13\u0026mu;lof each sample was transferred into new0.2ml PCR tubes either for immediate use or storage at -20℃\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRIP‑qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe endogenous RNA was captured in the nucleus or cytoplasm through antibody or epitope labeling, followed by isolation of RNA-binding proteins from the bound RNA using immunoprecipitation. Cells were crosslinked with 1% formaldehyde and treated with RIPA buffer containing 150 mM NaCl, RNase, and protease inhibitors. Subsequently, cell lysis was performed using a solution consisting of 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40, 1 mM EDTA, and 50 mM Tris (pH 8.0) for a duration of 30 minutes before centrifugation to collect the precipitates. The supernatant was then incubated four times with primary antibodies against METTL3 and YTHDF2 (or corresponding IgG antibodies), followed by addition of protein A/G glycosylated microspheres which were shaken for two hours. After washing the cells three times with RIPA buffer, RNA extraction was carried out following crosslinking procedures. Finally, quantitative RT-PCR was employed to detect the extracted RNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA pull‑down assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe initial step involved the utilization of the MEGAscript T7 Transcription Kit from Thermo Scientific to transcribe the RNA. Subsequently, we employed the Pierce RNA 3\u0026prime; End Desthiobiotinylation Kit (20, 163, Thermo Scientific) to label the ends of the amplified RNA with desthiobiotin. Lastly, we conducted RNA pulldown experiments using the Pierce Magnetic RNA\u0026ndash;Protein Pull-Down Kit (20, 164, Thermo Scientific). Specifically, a mixture was prepared by combining 2 mg of protein lysates, 50 pmol of biotinylated RNAs, and 50 \u0026micro;L of streptavidin beads. Following three washing cycles and an incubation period, immunoblotting analysis was performed after subjecting the streptavidin beads to boiling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLuciferase reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe regions of HMGB1 mRNA containing the methylation sites of METTL3 were cloned into a pGL3 plasmid. The mutated sequence was synthesized by GenePharma, located in Shanghai, China. To quantify luciferase activity, we utilized the dual-luciferase reporter assay system from Promega based in the United States. Using a GloMax 20/20 Luminometer also provided by Promega, we determined the relative luciferase activity as indicated by the ratio between firefly luciferase activity and Renilla luciferase activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical (IHC) staining \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor clinical specimens and mouse xenograft samples, paraffin embedding was carried out using the R.T.U. Vectastain Kit (from Vector Laboratories). Immunohistochemical staining was performed according to the instructions provided with the immunohistochemistry kit. All stainings were evaluated using quantitative imaging methods, where the percentage and intensity of immunostaining were recorded. The H-score was calculated using the following formula:\u003c/p\u003e\n\u003cp\u003eH-score = \u0026Sigma; (PI \u0026times; I)\u003c/p\u003e\n\u003cp\u003e= (Percentage of cells with weak intensity \u0026times; 1) + (Percentage of cells with moderate intensity \u0026times; 2) + (Percentage of cells with strong intensity \u0026times; 3)\u003c/p\u003e\n\u003cp\u003eHere, PI represents the percentage of positively stained cells among all cells, and I denotes the staining intensity.\u003c/p\u003e\n\u003cp\u003eKi-67 (Proteintech: 27309-1-AP, China); anti-CD133 (Proteintech: 66666-1-Ig, China)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid Construction and Lentiviral infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll plasmids were designed to clone the corresponding cDNA into the corresponding expression vector to produce the desired protein. For shRNA and PCDH vectors, the culture supernatant of HEK 293T cells was collected 48-72 hours after transfection for virus preparation and target cells were infected with 70% infection efficiency. PCDH lentivirus was used to construct METTL3 wild-type (WT) overexpression cell lines, while lentiCRISPR method was used to generate METTL3 knockout (KO) cell lines. In brief, we used BsmBI enzyme to linearize the lentiviral CRISPR vector and constructed the guide RNA (shRNA) into the lentiCRISPR V2 lentiviral expression vector. The shMETTL3 sequence: 5\u0026apos;-GCACTTGGATCTACGGAATCC-3\u0026apos;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProliferation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cellular proliferation was assessed using the EdU kit (provided by KeyGen Biotech Co., Ltd.) to quantify the EdU incorporation. After 48 hours of transfection, cells were seeded at a density of 2 \u0026times; 105 cells per well in a 6-well plate and exposed to 10 nM EdU for a duration of 12 hours. Subsequently, the cells were fixed and permeabilized with 0.5% Triton X-100 for a period of 20 minutes. Following this, the Click-iT reaction cocktail was introduced, and the cells were incubated under dark conditions for approximately half an hour. After two washes with PBS, DAPI dye was applied as a counterstain for about ten minutes in darkness before observing them using fluorescence microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSphere-forming assay \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PC cells were cultured in serum-free DMEM/F12 medium supplemented with 2% B-27 (Gibco, USA, #17504044), heparin (4 \u0026mu;g/ml), epidermal growth factor (20 ng/ml) (R\u0026amp;D, USA, #236-EG-200), and fibroblast growth factor (20 ng/ml) (PEPROTECH, USA, #AF-100-18C). DMEM/F12 medium with varying glucose concentrations was prepared by combining equal volumes of low-glucose DMEM medium (Gibco, USA) and F12 medium (Gibco, USA) containing d-glucose from Aladdin Industrial Corporation (#G116304). The cells were seeded at a density of 500 cells per well in ultra-low adhesion 6-well plates. After 7 days of incubation, the cultures were examined for sphericity using a phase contrast optical microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo perform staining, a total of 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were seeded into a U-shaped 96-well plate and incubated with 5 \u0026micro;l of each antibody at a temperature of 4\u0026deg;C for a duration of 30 minutes. Following PBS washing, the cells were collected by centrifugation at a gravity force of 1000 g for 5 minutes. Subsequently, the cells were resuspended in 300 \u0026micro;l of PBS and subjected to flow cytometry analysis. The CD133/1-PE antibody was procured from MiltenyiBiotec located in Bergisch Gladbach, Germany.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial superoxides measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MitoSOX\u0026trade; Red mitochondrial superoxide indicator for live-cell imaging (Invitrogen) was utilized to measure the accumulation of mitochondrial superoxide. The manufacturer\u0026apos;s protocol was followed for this purpose. Briefly, adherent cells were cultured on glass coverslips in a six-well plate. After the specified treatments, a 5 \u0026mu;M MitoSOX\u0026trade; working solution (1 ml) was added to the cells on the coverslips. Subsequently, incubation of the cells at 37\u0026deg;C in darkness took place for 10 minutes. Following this, gentle washing with warm buffer was performed three times. Mounting of coverslips in warm buffer facilitated imaging using a confocal microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of intracellular ROS, malondialdehyde (MDA), and glutathione (GSH)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CellROX\u0026trade; Deep Green Reagent (Invitrogen, C10444) is employed in the experimental procedure to observe fluorescence microscopy and detect intracellular levels of ROS. Briefly, prior to treatment, samples are exposed to a 5 \u0026mu;M solution of the reagent for a duration of 30 minutes. Subsequently, after a post-treatment period lasting 0.5 hours, cells are gathered for measurements of fluorescence intensity. The evaluation of Malondialdehyde (MDA) and Glutathione (GSH) production within the cells is conducted using MDA Detection Kit and GSH Assay Kit respectively, which are provided by Solarbio Science \u0026amp; Technology Co., Ltd., Beijing, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial membrane potential measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTetramethylrhodamine Ethyl Ester (TMRE) (manufactured by Beyotime) is a commonly employed fluorescent dye for labeling mitochondria in viable cells. As per the provided guidelines, an appropriate quantity of TMRE buffer is introduced to the cells, which are then incubated at 37\u0026deg;C in a light-restricted environment for a duration of 20 minutes. Following this, the cells are rinsed with serum-free medium and subsequently observed using a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntracellular iron assay \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentration of ferrous ions within the cell was assessed by utilizing FerroOrange dye (#F374, Dojindo Laboratories). Cells were cultured and subjected to specific treatments. Subsequently, a diluted solution (1:2000, v/v) of FerroOrange Orange dye was added to the cells and incubated at 37\u0026deg;C for 30 minutes. Fluorescent images were captured using a confocal microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoprecipitation (IP) and Co-immunoprecipitation (co-IP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were disrupted in Pierce IP buffer (Thermo Fisher) supplemented with protease and phosphatase inhibitors (Sigma). Following incubation with specified antibodies, the lysates were combined with protein A/G agarose beads (Thermo Fisher). For proteins carrying a tag, we employed immunomagnetic beads conjugated to anti-Flag/anti-HA antibody. Subsequent to immunoprecipitation, protein A/G agarose or magnetic beads underwent three washes using TBST (0.1% Tween-20, 150 mM NaCl, 10 mM Tris-HCl pH7.5), followed by elution in SDS lysis buffer (100 mM NaCl, 1% SDS, 50 mM Tris-HCl pH 7.5) for western blot analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo ubiquitination assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn ubiquitination experiments, cells were transiently transfected with the specified plasmids for 36 hours, followed by treatment with 20 \u0026mu;M MG132 for an additional 8 hours. The cells were then harvested. One-fifth of the cells were reserved for direct immunoblotting, while the remainder were processed for denaturing co-immunoprecipitation. The cells were lysed in denaturing buffer (described by Liu et al., 2023), and the lysates were incubated overnight HA-beads (Millipore; IP0010) at 4\u0026deg;C. Subsequently, the beads were washed with buffers and Buffer C. Finally, the immunocomplexes were eluted using elution buffer, and the samples were analyzed by immunoblotting with indicated antibodies as detailed by Liu et al\u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXenograft Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eour-week-old nude mice were purchased from Cavens, Changzhou, China. The nude mice were randomly allocated into 5 groups without any specific selection criteria. The mice were raised under specific pathogen-free conditions. Pancreatic cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) stably expressing METTL3 and HMGB1 were implanted into the right flank of each mouse and allowed to grow, with 6 mice in each group (n = 6). Six days after cancer cell injection, gemcitabine (120 mg/kg, Sigma-Aldrich) was administered weekly for the gemcitabine treatment cohort, while RSL3 (5 mg/kg, Sigma-Aldrich) was injected once daily for the combined gemcitabine and RSL3 treatment group. Both gemcitabine and RSL3 were administered via intraperitoneal injection. This treatment regimen continued until the final observation week. The animal experimental protocol was approved by the Animal Ethics Committee of the Animal Care and Use Committee of the Ethical Institution of The Second Affiliated Hospital of Kunming Medical University. The investigators did not blind the animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe survival curve of METTL3 in pancreatic cancer was obtained from the database https://smuonco.shinyapps.io/PanCanSurvPlot/; METTL3 differential genes and METTL3 peak genes were obtained from GSE146806 and GSE132306; METTL3 targeted genes were downloaded from the database http://rm2target.canceromics.org/#/home, and METTL3 differential genes in pancreatic cancer were obtained from UALCAN (uab.edu); ferroptosis-related genes were obtained from FerrDb (zhounan.org); the expression of related proteins such as METTL3 and YTHDF2 in pancreatic cancer was downloaded from Proteomic Data Commons (cancer.gov); Welcome to SRAMP, an online m6A site predictor (cuilab.cn) predicted HMGB1 m6A modification sites; the docking model of HMGB1 and gemcitabine was analyzed by autodockvina and visualized by Pymol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were collected from a minimum of three independent experiments. Statistical analysis was conducted using Prism software (GraphPad Prism 9.0.0). Unless otherwise stated, all data are presented as mean \u0026plusmn; standard deviation. Paired or unpaired Student\u0026apos;s t-test (two-tailed) was employed for experiments involving two groups only. One-way ANOVA with multiple comparisons was utilized for experiments with more than two groups. Two-way ANOVA with multiple comparisons was applied for comparing four or more groups in a two-factor experiment. The level of statistical significance was set at *p\u0026lt;0.05, ** p\u0026lt;0.01 to determine significant differences among the experimental groups.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eMETTL3 is upregulated in pancreatic cancer and promotes pancreatic cancer cell proliferation, stemness, and gemcitabine resistance\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the impact of METTL3 expression on the prognosis of pancreatic cancer patients, we utilized the PanCanSurvPlot database and observed that patients with high METTL3 expression exhibited a poor prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We collected multiple samples of pancreatic cancer tissues along with adjacent normal tissues and detected an upregulation in METTL3 expression in tumor tissues through qPCR analysis (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Furthermore, immunohistochemistry and Western blot analyses confirmed a significant upregulation of METTL3 expression in tumor tissues compared to normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). In order to clarify the role of METTL3 in the progression of pancreatic cancer, we established stable knockdown and overexpression models of METTL3 in two pancreatic cancer cell lines: PACN1 and BXCP3 (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Utilizing the CCK8 assay, we discovered that overexpression of METTL3 promoted proliferation in both PACN1 and BXCP3 cells, while knockdown of METTL3 inhibited their growth (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC, D). Furthermore, we observed a decrease in the number of EdU-positive cells in PACN1 and BXCP3 upon knockdown of METTL3 compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Gemcitabine is currently considered as the first-line treatment for pancreatic cancer; however, resistance to gemcitabine has emerged as a major obstacle. Interestingly, our findings indicate that lower levels of METTL3 exhibit greater sensitivity to gemcitabine compared to higher levels of METTL3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F). Given that cell pluripotency has been linked to GEM resistance \u003csup\u003e32\u003c/sup\u003e, we further investigated the impact of METTL3 on pancreatic cancer stemness. Initially, we conducted cell sphere formation experiments and observed a significant reduction in both volume of spheres formed by PANC1 and BXPC3 cells following METTL3 knockout (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG; Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE). Additionally, flow cytometry analysis revealed a notable decrease in the percentage of CD133\u0026thinsp;+\u0026thinsp;cells - a marker for stemness - upon METTL3 knockout in PANC1 and BXPC3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH; Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe inhibition of ferroptosis in pancreatic cancer cells is mediated by METTL3\u003c/h3\u003e\n\u003cp\u003eTo further elucidate the molecular mechanism underlying METTL3-mediated regulation of pancreatic cancer progression, we retrieved the GSE146806 and GSE132306 datasets from the GEO database. Subsequently, a KEGG analysis was performed on the overlapping genes between the top 2,000 differentially expressed genes identified in GSE146806 and the genes corresponding to METTL3 binding RNA peaks in GSE13,306. Our findings revealed that METTL3 is involved in regulating ferroptosis (Fig \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). Consequently, knockdown of METTL3 in stable knockout and stable overexpression models of PANC1 and BXPC3 led to an elevation in mitochondrial superoxide levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Fig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA), increased MDA production (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Fig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB), reduced GSH levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Fig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC), accompanied by augmented ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, Fig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD) and iron ion concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, Fig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eE), as well as decreased mitochondrial membrane potential levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, Fig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF). Conversely, overexpression of METTL resulted in an opposite phenotype. Collectively, these results indicate that METTL inhibits cell ferroptosis and consequently promotes pancreatic cancer progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eHMGB1 was identified as a downstream target of METTL3\u003c/h2\u003e \u003cp\u003eAs METTL3 is an M6A methyltransferase, we firstly detected the effect of METTL3 on the overall m6A level in PANC1 and BXPC3 cells. The results showed that METTL3 significantly increased the overall m6A level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). The above experiments have shown that METTL3 regulates ferroptosis in pancreatic cancer cells. In order to determine the downstream ferroptosis target regulated by METTL3, we used a Venn diagram showing differential genes obtained from GSE146806 analysis, correlation genes of METTL3 in pancreatic cancer obtained from UALCAN, target genes from RM2Target database, and the genes corresponding to METTL3 binding RNA peaks in GSE13306 and ferroptosis-related genes to obtain HMGB1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The RIP-qPCR assay was employed to validate the binding of METTL3 in PDAC cells with either knockdown or overexpression. Overexpression of METTL3 led to an increased enrichment of HMGB1, whereas knockdown of METTL3 resulted in a significant decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB). RNA pull-down analysis demonstrated the direct interaction between full-length HMGB1 mRNA and the METTL3 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). MeRIP-qPCR results revealed that the presence of METTL3 directly enhanced m6A modification on HMGB1 mRNA in PANC1 and BXPC3 cells (Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eC, D). Furthermore, Western blotting showed that depletion of METTL3 elevated HMGB1 protein levels, while overexpression of METTL3 reduced them (Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eE), which was further confirmed by qPCR analysis (Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eF). Additionally, actinomycin D treatment was performed to assess the impact of METTL3 on HMGB1 mRNA stability, demonstrating that knockdown of METTL3 resulted in increased stability for HMGB1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). We referred to the SRMAP database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cuilab.cn/sramp\u003c/span\u003e\u003cspan address=\"http://www.cuilab.cn/sramp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to determine the m6A sites, and found two highly confident m6A sites in the 3' UTR. Next, we conducted a luciferase assay by constructing the 3' UTR of HMGB1 (Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eG) to evaluate the effect of METTL3 binding on transcription. In the mutant group, the adenine nucleotide at the m6A site was replaced with cytosine to eliminate the m6A modification. The relative luciferase activity (Firefly/ Renilla ratio) ) showed that METTL3 silencing enhanced the transcriptional activity of the 3' UTR of HMGB1. It is noteworthy that the mutation at position 1535 eliminated the effect of METTL3 silencing, while the mutation at position 791 did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). The MeRIP-qPCR results further confirmed that position 1535 mediated the m6A methylation of HMGB1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eH) and the stability of HMGB1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eYTHDF2 mediates HMGB1 mRNA expression in an m6A-dependent manner\u003c/h2\u003e \u003cp\u003eThe dynamic and reversible regulation of m6A modification is determined by the interaction between m6A writers and erasers. However, different downstream biological functions necessitate the recognition of m6A by distinct readers, including the regulation of m6A-modified transcripts. Stability analysis of HMGB1 mRNA revealed that METTL3 deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F) and mutation at the m6A site on HMGB1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eI) both enhanced mRNA stability. Previous studies have reported that YTHDF2 recognizes m6A modification to promote degradation of its target gene mRNA\u003csup\u003e33\u003c/sup\u003e. Through RNA pull-down experiments and western blotting, we identified YTHDF2 as an m6A reader for HMGB1 mRNA in PANC1 and BXPC3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). qPCR experiments demonstrated a negative correlation between YTHDF2 expression and HMGB1 mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), which was further confirmed by western blotting showing increased levels of HMGB1 protein upon knocking down YTHDF2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Analysis of protein expression data from the Proteomic Data Commons database revealed a negative correlation between YTHDF2 and HMGB1 protein expression in pancreatic cancer samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Additionally, RIP-qPCR experiments showed that knocking down YTHDF2 led to increased levels of HMGB1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Relative luciferase activity assays indicated that silencing YTHDF2 enhanced luciferase activity associated with HMGB1, while mutation at site 1535 abolished this effect caused by YTHDF2 silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), providing further evidence for the role of knocking down YTHDF2 in enhancing stability of HMGB1 mRNA in PANC-1 and BXPC-3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Our findings suggest a mechanistic control exerted by YTHDF2 on stability and expression levels of HMGB1 mRNA through an m6A-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eInhibition of ferroptosis by METTL3 and gemcitabine resistance are mitigated by HMGB1\u003c/h2\u003e \u003cp\u003eOur previous studies have demonstrated that METTL3 inhibits ferroptosis and induces resistance to gemcitabine. HMGB1, a downstream target of METTL3 in the context of ferroptosis, the association between ferroptosis and gemcitabine resistance has been extensively documented in the literature. To investigate whether HMGB1 can regulate the sensitivity of pancreatic cancer cells to gemcitabine, we initially employed autodock vina software for analyzing the free energy between HMGB1 and gemcitabine molecules. The complexing free energy between HMGB1 and GEM was found to be -6.1 kcal/mol. Additionally, we visualized the docking model using pymol software (Fig \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA), suggesting that HMGB1 may serve as a downstream molecular target of GEM. We evaluated the impact of HMGB1 on GEM sensitivity in PANC1 and BXPC3 cells overexpressing METTL3. Our findings revealed that HMGB1 could enhance the sensitivity of METTL3-overexpressing cells towards GEM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Similarly, HMGB1 could mitigate the effect of METTL3 on cell stemness by reducing sphere-forming cell diamater (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and CD133\u003csup\u003e+\u003c/sup\u003e cell populations (Fig \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eB). Subsequently, we investigated whether HMGB1 could alleviate the inhibition of ferroptosis caused by METTL3 in pancreatic cancer cells. For non-transfected cells, transfection with HMGB significantly reduced ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and MDA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), while upregulating GSH levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These results suggest that through alleviating ferroptosis inhibition and gemcitabine resistance induced by METTL3 overexpression, HMGB contributes to cellular responses in pancreatic cancer cells. To explore synergistic effects among gemcitabine treatment, ferroptosis induction, and HMBG expression on cell proliferation rates; we first assessed cell viability revealing that overexpression of HMBG combined with treatment involving both gemcitabine and RSL3 significantly reduced cell viability rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, G). The combined treatment of HMGB1 and GEM with RSL3 significantly suppressed tumor proliferation in in vivo experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, Fig \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eC, D), immunohistochemistry also confirmed this result (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). These findings suggest that targeting HMGB1 in conjunction with GEM and ferroptosis represents a promising therapeutic strategy for pancreatic cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eO-GlcNAcylation stabilizes METTL3 protein via enhancing the interaction between METTL3 and EIF3H\u003c/h2\u003e \u003cp\u003eOur previous studies have demonstrated that high expression of METTL3 is associated with a poor prognosis in pancreatic cancer and promotes the progression of pancreatic cancer cells. In order to identify new molecular mechanisms regulating the function of METTL3, we discovered its involvement in lipid metabolism, central carbon metabolism, amino acid metabolism, and nucleic acid metabolism (Fig \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). Additionally, O-GlcNAcylation integrates glucose, amino acids, fatty acids, and nucleic acid metabolism \u003csup\u003e15\u003c/sup\u003e. As shown in the UALCAN database, OGT also shows a significant upregulation of protein expression in pancreatic cancer (Fig \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA). Therefore, we hypothesized whether METTL3 undergoes O-GlcNAcylation mediated by OGT (O-GlcNAc Transferase). Initially, we obtained the 3D structure of the METTL3-METTL14 complex and OGT dimer from the PDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), as well as the complex model of METTL3-METTL4 and OGT from Cluspro (Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eA). The binding free energy analysis using \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e revealed that the binding free energy between METTL3-OGT is less than 0༈Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eB༉, suggesting an interaction between them. Co-IP experiment confirmed this interaction between METTL3 and OGT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Furthermore, while the K908A mutation in OGT eliminated its enzymatic function without affecting protein abundance according to previous research\u003csup\u003e34\u003c/sup\u003e, our study found that this mutant could still interact with METTL3. However, this mutant failed to induce O-GlcNAcylation of METTL3, suggesting that enzymatic activity of OGT is necessary for the process (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To determine the region where OGT and METTL3 interact, we constructed a truncated model of OGT (Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eC). We found that, except for the δTPR region, METTL3 could bind to it (Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eD), indicating that METTL3 interacts with the OGT-TPR region. Since O-GlcNAcylation usually affects protein stability, we downloaded the expression of METTL3 and OGT proteins in pancreatic cancer patients from the Proteomic Data Commons and plotted a correlation curve, which showed that OGT protein expression and METTL3 protein expression were positively correlated in pancreatic cancer (Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eE). In PANC1 cells, we detected the expression of METTL3 and the overall O-GlcNAcylation by interfering with OGT and giving PugNAc to inhibit OGA, and found that after inhibiting OGT, the expression level of METTL3 protein was lower, while inhibiting OGA could enhance METTL3 protein expression, and the expression of METTL3 was significantly downregulated after O-GlcNAcylation inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C). However, O-GlcNAcylation did not affect the expression of METTL3 mRNA (Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eF). Next, we detected the effect of O-GlcNAcylation on the stability of METTL3 protein. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, knocking down OGT and inhibiting O-GlcNAcylation promoted the degradation of METTL3. The ubiquitin-proteasome pathway mediates 80%-85% of protein degradation\u003csup\u003e35\u003c/sup\u003e. O-GlcNAcylation can prevent the degradation of target proteins by reducing their ubiquitination, and the underlying mechanism is to recruit deubiquitinating enzymes to O-GlcNAcylated proteins\u003csup\u003e16\u003c/sup\u003e. The ubiquitination test also confirmed that knockdown of OGT and K908A mutations did enhance the level of METTL3 ubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), further clarifying that OGT mainly mediates polyubiquitination in a K48-dependent manner (Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eG). Next, we explored the mechanism by which O-GlcNAcylation regulates the stability of METTL3. We downloaded the binding protein of METTL3 (PXD036899) from the proteomexchange.org database, downloaded the interacting proteins of METTL3 and all deubiquitinating enzymes from the databases \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://thebiogrid.org/\u003c/span\u003e\u003cspan address=\"https://thebiogrid.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://iuucd.biocuckoo.org/index.php\u003c/span\u003e\u003cspan address=\"https://iuucd.biocuckoo.org/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e respectively, and obtained the only overlapping protein EIF3H through the Venn diagram (Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eH). We verified that there was no correlation between EIF3H mRNA and METTL3 mRNA in the GEPIA database (Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eI), but a positive correlation at the protein level (Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eJ). The above data illustrate that EIF3H regulates the expression of METTL3 protein. The ubiquitination experiment demonstrated that knockdown of EIF3H significantly enhanced the ubiquitination of METTL3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Through Co-IP experimentation, we observed a binding interaction between EIF3H and METTL3(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). By analyzing the binding free energy of EIF3 and METTL3, as well as EIF3 and METTL3-OGT complexes using Cluspro, we discovered that the binding free energy of EIF3 and METTL3-OGT complexes was lower compared to that of EIF3 and METTL3 within a similar interaction area. This suggests that OGT has the ability to enhance the binding affinity between METTL3 and EIF3 (Fig \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eB). The Co-IP results further confirmed this observation, demonstrating that the addition of OGT augmented the interaction between METTL3 and EIFH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Similarly, PugNAc treatment also enhanced the binding between METTL3 and EIF3H (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). We combined multiple databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/NetOGlyc-4.0/;https://services.healthtech.dtu.dk/services/DictyOGlyc-1.1/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/NetOGlyc-4.0/;https://services.healthtech.dtu.dk/services/DictyOGlyc-1.1/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ༛\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.oglcnac.mcw.edu/\u003c/span\u003e\u003cspan address=\"https://www.oglcnac.mcw.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ༉ to predict that the O-GlcNAcylation modification site of METTL3 is S118, and this site is highly conserved in mice, rats and humans. We produced a mutant of METTL3 (S118A). The IP assay also showed significantly lower levels of O-GlcNAcylation on S118A compared to WT (Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eK), indicating that S118 is the main site of O-GlcNAcylation of METTL3. Compared with WT, the S118A mutant promoted the degradation of METTL3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI), inhibited the binding of METTL3 and EIF3H (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ), and the ubiquitination experiment also proved that the S118A mutation significantly enhanced the ubiquitination modification of METTL3 compared with WT(Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eL). The findings suggest that O-GlcNAcylation promotes the stability of METTL3 by enhancing the interaction between METTL3 and EIF3H.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eMore and more evidence suggests that M6A modification plays a crucial role in various biological processes, especially in tumor formation and cancer progression. As the catalytic subunit of the M6A writer, METTL3 has been found to collaborate with YTHDF2 and promote HCC progression by degrading the mRNA of SOCS2 \u003csup\u003e27\u003c/sup\u003e. In pancreatic cancer, it has also been proven that METTL3 modulates ID2 by m6A methylation to regulate pancreatic cancer cell stemness\u003csup\u003e36\u003c/sup\u003e. Previous studies mainly focused on the role of METTL3 in mediating mRNA metabolism and tumor progression through its target gene m6A methylation regulation, however, the limited understanding of METTL3-specific functional regulation, especially in post-translational modification, still exists. Here, we first prove that METTL3 is highly expressed in pancreatic cancer and inhibits ferroptosis-induced gemcitabine resistance, and HMGB1 is identified as a downstream target of METTL3. Furthermore, the m6A-YTHDF2-dependent pathway is downregulated to decrease HMGB1. Targeting HMGB1, combined with gemcitabine and ferroptosis inducer RSL3, can significantly inhibit tumor proliferation. Furthermore, we prove that METTL3 exists in O-GlcNAcylation, and stabilizes its expression, the S118 site O-GlcNAcylation maintains the stability of the METTL3 protein, the specific mechanism lies in that O-GlcNAcylation interferes with ubiquitination, i.e., O-GlcNAcylation enhances the interaction between METTL3 and deubiquitinase EIF3H, resulting in a lower level of ubiquitination modification and enhanced stability of the METTL3 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). These findings provide new insights into the treatment of pancreatic cancer.\u003c/p\u003e \u003cp\u003eIn recent years, post-translational modification of METTL3 has attracted more and more attention from academia. SUMOylation of METTL3 at K177, K211, K212 and K215 sites might repress its methyltransferase activity for m6A RNA methylation. Subsequent SUMOylation of METTL3 promotes colony formation and tumor growth of human non-small cell lung cancer (NSCLC) H1299 cells\u003csup\u003e37\u003c/sup\u003e, which is caused by downstream gene dysregulation. Lacylation-driven METTL3-mediated RNA m6A modification plays an important role in promoting the immunosuppressive ability of tumor-infiltrating myeloid cells\u003csup\u003e38\u003c/sup\u003e. In addition, METTL3 can bind to USP5, and this binding is promoted by ERK-mediated phosphorylation. ERK-dependent METTL3 stabilization affects cellular mRNA m6A methylation which could contribute to tumorigenesis\u003csup\u003e39\u003c/sup\u003e. The E3 ubiquitin ligase RNF113A mediates the ubiquitin/proteasome-dependent degradation of METTL3 through the K48-linked multi-ubiquitin chain, reducing the level of m6A modification to promote the inhibition of acute myeloid leukemia\u003csup\u003e40\u003c/sup\u003e. Our study revealed that OGT induced O-GlcNAcylation of METTL3 by interacting with METTL3, increasing the stability of METTL3 protein. In addition, we also found that METTL3 was O-GlcNAcylated at Ser118 site and emphasized its role in crosstalk ubiquitination. The improvement of METTL3 stability led to the enhancement of the level of m6A modification of downstream target genes. These findings also further clarify the regulatory network of post-translational modification of proteins and m6A modification of RNA in the pathogenesis of pancreatic cancer.\u003c/p\u003e \u003cp\u003eO-GlcNAcylation has been demonstrated to impact various functional activities of proteins, including stability, transcriptional activity, localization, and protein-protein interactions \u003csup\u003e17\u003c/sup\u003e. The presence of O-GlcNAcylation at Thr58 competes with phosphorylation, leading to enhanced protein stability and hindered degradation by proteasomes for c-MYC \u003csup\u003e41\u003c/sup\u003e. Inhibition of O-GlcNAcase (OGA) activity results in prolonged half-life of YAP through the inhibition of SCF\u003csup\u003eβ\u0026minus;TRCP\u003c/sup\u003e E3-ubiquitin ligase \u003csup\u003e42\u003c/sup\u003e. Moreover, accumulating evidence suggests that elevated levels of O-GlcNAcylation are closely associated with pancreatic cancer progression, metastasis and recurrence, ECM remodeling, and immunotherapy resistance\u003csup\u003e43,44\u003c/sup\u003e.In our study on pancreatic cancer, we observed a regulatory relationship between METTL3 expression and the extent of O-GlcNAcylation modification. Specifically, enhancing O-GlcNAcylation of METTL3 through exogenous transfection of OGT increased its protein stability. Conversely, loss-of-function mutation in the catalytic domain of OGT (K908A) reduced METTL3 expression. Similarly, treatment with PugNAc (an inhibitor targeting OGA) upregulated METTL3 expression. Deficiency in O-GlcNAcylation shortened the half-life of METTL3 by promoting K48-linked ubiquitination. Additionally, loss-of-function mutation S118A resulted in enhanced stability of METTL3 by inhibiting its binding to EIF3H protein via abrogating the interaction with this partner molecule. Further investigations are required to determine whether degradation-induced regulation downstream phenotypes occur due to loss-of-O-GlcNAcylation caused by S118A mutation. Interestingly, the S118A mutant does not completely abolish O-GlcNAcylation of METTL3, thereby implying the existence of potential alternative O-GlcNAc sites.\u003c/p\u003e \u003cp\u003eHigh mobility group box 1 (HMGB1) is a non-histone chromatin-associated protein that is widely distributed in eukaryotic cells and plays a role in DNA damage repair and genomic stability maintenance \u003csup\u003e45\u003c/sup\u003e. HMGB1 appears to have conflicting functions in the progression and treatment of cancer. On the one hand, HMGB1 may promote tumor formation, for example, by playing an important role in regulating mouse oval cell activation and liver cancer development related to inflammation\u003csup\u003e46\u003c/sup\u003e. On the other hand, LPS induces pro-inflammatory cytokines (such as IL-1β, IL-6, and TNF-α) in a HMGB1-dependent manner to improve colorectal cancer progression \u003csup\u003e47\u003c/sup\u003e. In terms of resistance, both the nuclear and cytoplasmic HMGB1 promote autophagy and inhibit tumor cell apoptosis to induce chemotherapy resistance\u003csup\u003e48\u003c/sup\u003e. The role of HMGB1 in ferroptosis is also increasingly reported. Neutrophil extracellular traps mediate cardiomyocyte ferroptosis via the Hippo-Yap pathway to exacerbate doxorubicin-induced cardiotoxicity\u003csup\u003e49\u003c/sup\u003e, where HMGB1 acts as an iron apoptosis inducer by upregulating ferroptosis in astrocytes, thereby aggravating the acute injury after ischemia in the brain \u003csup\u003e50\u003c/sup\u003e. In pancreatic ductal adenocarcinoma, Targeting the MCP-GPX4/HMGB1 Axis for Effectively Triggering Immunogenic Ferroptosis\u003csup\u003e13\u003c/sup\u003e; N6F11 treatment caused ferroptotic cancer cell death that initiated HMGB1-dependent antitumor immunity mediated by CD8\u0026thinsp;+\u0026thinsp;T cells\u003csup\u003e51\u003c/sup\u003e. Previous studies have shown that post-translational modifications (i.e., acetylation, phosphorylation, and methylation) of HMGB1 near or within its nuclear localization sequences (NLS) can induce its translocation to the cytoplasm, leading to the subsequent release of HMGB1 during inflammation \u003csup\u003e52\u0026ndash;54\u003c/sup\u003e. In sepsis, YTHDF2 inhibits the release of HMGB1 and alleviates inflammatory responses\u003csup\u003e55\u003c/sup\u003e, but no further exploration has been made on the mechanism of YTHDF2 regulating HMGB1 expression; in primary liver cancer, HMGB1 is demethylated by ALKBH5 and recognized by Reader YTHDF2, promoting its degradation\u003csup\u003e56\u003c/sup\u003e. Here, we demonstrate for the first time that HMGB1 is a target gene of METTL3 and is degraded by METTL3 in a m6A-YTHDF2-dependent manner. Furthermore, we found that the supplementation of HMGB1 could alleviate the inhibition of ferroptosis by METTL3 in pancreatic cancer. Our study reveals that HMGB1 is a target protein of Writer METTL3 and proves that METTL3 regulates HMGB1 in a m6A-dependent manner, thereby regulating ferroptosis. This provides a certain basis for a new treatment direction in pancreatic cancer.\u003c/p\u003e \u003cp\u003eIn summary, we have elucidated the molecular mechanism underlying METTL3-mediated regulation of ferroptosis and gemcitabine resistance in pancreatic cancer. Our findings demonstrate that elevated expression of METTL3 in pancreatic cancer is partially dependent on O-GlcNAcylation, which is mediated by OGT. The stabilization of METTL3 protein expression through O-GlcNAcylation at the S118 site enhances its interaction with deubiquitinase EIF3H. Moreover, we have discovered that METTL3 promotes the degradation of ferroptosis driver HMGB1 in an m6A-YTHDF2-dependent manner. Additionally, our study reveals that a combination therapy targeting HMGB1, along with gemcitabine and the ferroptosis inducer RSL3, effectively suppresses pancreatic cancer development. However, our study has certain limitations including insufficient exploration of downstream mechanisms involved in regulating ferroptosis and gemcitabine resistance upon O-GlcNAcylation-induced increase in METTL3 stability. Furthermore, clinical data supporting these findings are lacking in our dataset. In future studies, we aim to investigate whether O-GlcNAcylation of METTL3 can regulate its resistance to ferroptosis and gemcitabine treatment while also exploring its impact on the tumor microenvironment and potential modulation of immune checkpoint blockade efficacy through an m6A-dependent mechanism. These efforts will provide valuable insights into the potential clinical application of METTL3 for treating pancreatic cancer and improving treatment outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical\u0026nbsp;and\u0026nbsp;Legal\u0026nbsp;Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study and included experimental procedures were approved by The Second Affiliated Hospital of Kunming Medical University. All animal experiments were approved by the Animal\u0026nbsp;Care\u0026nbsp;and\u0026nbsp;Use\u0026nbsp;Committee\u0026nbsp;of\u0026nbsp;the\u0026nbsp;Ethical\u0026nbsp;Institution\u0026nbsp;of The Second Affiliated Hospital of Kunming Medical University\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent\u0026nbsp;for\u0026nbsp;publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot\u0026nbsp;applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability\u0026nbsp;of\u0026nbsp;data\u0026nbsp;and\u0026nbsp;materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;datasets\u0026nbsp;used\u0026nbsp;and/or\u0026nbsp;analysed\u0026nbsp;during\u0026nbsp;the\u0026nbsp;current\u0026nbsp;study\u0026nbsp;are\u0026nbsp;available\u0026nbsp;from\u0026nbsp;the\u0026nbsp;corresponding\u0026nbsp;author\u0026nbsp;on\u0026nbsp;reasonable\u0026nbsp;request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting\u0026nbsp;interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;authors\u0026nbsp;confirm\u0026nbsp;that\u0026nbsp;there\u0026nbsp;are\u0026nbsp;no\u0026nbsp;conflicts\u0026nbsp;of\u0026nbsp;interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project is supported by Yunnan Provincial Applied Basic Research Plan (202401AT070020), Yunnan Provincial Science and Technology Department Basic Research Plan (202101AY070001-142), Kunkun-Medical joint special project number: 202301AY070001-270, 202101AY070001-145, Scientific Research Foundation of Education Department of Yunnan Province (2024J0346), Academician Expert Workstation of Yunnan Province (202205AF150127), Supported by Weichuang Treatment Innovation Team of Hepatobiliary and Pancreatic Surgery Department of Yunnan Province (202405AS350021)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026nbsp;contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQiuhongWang,\u0026nbsp;Dongyun Cun,\u0026nbsp;Dong Wei\u0026nbsp;and\u0026nbsp;Xiawei Yang made contribution to the conception and design;\u0026nbsp;Chunman Li, Kun Su,\u0026nbsp;Tao Wang,\u0026nbsp;Renchao Zou\u0026nbsp;and\u0026nbsp;Lianmin Wang\u0026nbsp;analyzed and interpreted data; QiuhongWang drafted the article;\u0026nbsp;Tao Wu, Bo Tang and Dongyun Cun\u0026nbsp;revisied it critically for important intellectual content; All authors approved the final version to be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBalachandran, V. 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Although ferroptosis is important in cancer therapy, the regulatory mechanism of METTL3 in pancreatic cancer and its potential for regulating ferroptosis as a treatment remain unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe relationship between METTL3 and gemcitabine resistance and ferroptosis was investigated through in vitro and in vivo functional gain and loss experiments, while the target genes of METTL3 were identified using siRNA and pharmacological inhibitors to analyze the impact of O-GlcNAcylation on METTL3's stability and ubiquitination modification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe found that O-GlcNAcylation interferes with ubiquitination-mediated regulation of METTL3 stability in pancreatic cancer cells, further clarifying its O-GlcNAcylation site and ubiquitination modification type. METTL3 regulates ferroptosis and Gemcitabine resistance by promoting HMGB1 degradation in a manner dependent on m6A-YTHDF2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eOur study findings clearly demonstrate that METTL3 promotes pancreatic cancer cell proliferation and drug resistance to gemcitabine. This highlights the role of O-GlcNAcylation in stabilizing METTL3 expression and degrading HMGB1 through a m6A-YTHDF2-dependent mechanism, thereby inhibiting ferroptosis in pancreatic cancer cells. Furthermore, targeting HMGB1 while coordinating gemcitabine and RSL3 significantly suppresses pancreatic cancer tumor growth. These results provide valuable insights for the treatment of pancreatic cancer.\u003c/p\u003e","manuscriptTitle":"O-GlcNAcylation with ubiquitination stabilizes METTL3, promoting HMGB1 degradation to inhibit ferroptosis and enhance gemcitabine resistance in pancreatic cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-17 04:14:08","doi":"10.21203/rs.3.rs-5606582/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5d38fd18-b0fe-4d29-b64c-52e2f78dab1b","owner":[],"postedDate":"December 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41595830,"name":"Health sciences/Oncology/Surgical oncology"},{"id":41595831,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Pancreatic cancer"}],"tags":[],"updatedAt":"2024-12-17T04:14:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-17 04:14:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5606582","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5606582","identity":"rs-5606582","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}