
METTL3‑mediated m6A methylation and its impact on OTUD1 expression in chronic obstructive pulmonary disease
- Authors:
- Published online on: May 19, 2025 https://doi.org/10.3892/mmr.2025.13571
- Article Number: 206
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Copyright: © Gao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
The Global Initiative for Chronic Obstructive Lung Disease defines chronic obstructive pulmonary disease (COPD) as ‘a common, preventable and treatable disease characterized by persistent airflow limitation due to airway and/or alveolar abnormalities, typically caused by significant exposure to toxic particles or gases’ (1). The main pathological features of COPD include chronic and abnormal inflammatory responses mediated by the NF-κB signaling cascade in the lungs (2) and epithelial-mesenchymal transition (EMT) (3). As the third leading cause of death worldwide (4), the global mortality rate of COPD remains high, especially with the growing global population and increasing life expectancy. Moreover, the COVID-19 pandemic has notably impacted the quality of care and experience for patients with COPD (5). Some studies have shown significantly increased mortality in patients with COPD infected with COVID-19 (up to 32.2%) (5,6). Therefore, there is a need to explore timely diagnostic and therapeutic approaches for COPD.
COPD is often considered a smoking-induced disease, with smoking (including secondhand smoke) being the most notable risk factor. A total of ~1.5 million of the 3 million global deaths from COPD each year are attributed to smoking (7), and 8 million individuals die annually from smoking-related diseases (8). Other factors associated with COPD include air pollution (9), occupational exposure (10), environmental tobacco smoke (11), infections and low socioeconomic status (12). Patients with advanced COPD often succumb to severe respiratory distress and acute exacerbations. Current treatments, such as bronchodilators and anti-inflammatory drugs, focus on symptom relief and exacerbation management rather than targeting disease progression and mortality, with the underlying mechanisms of inflammation in COPD development and treatment remaining unclear (13). Thus, the exploration into COPD mechanisms and the development of novel therapies are crucial, with an urgent need to develop treatments targeting immune dysfunction and underlying inflammation to slow COPD progression.
Ovarian tumor protease (OTU) domain-containing protein 1 (OTUD1) is a member of the OTU subfamily of deubiquitinating enzymes with an N-terminal disordered alanine, proline, glycine-rich region, catalytic OTU domain and ubiquitin-interacting motif. OTUD1 can cleave various ubiquitin linkages and is involved in regulating multiple cellular functions (14). Studies have shown that OTUD1 inhibits the progression of non-small cell lung cancer by mediating KLF4 stabilization, and that the deubiquitinase OTUD1, upregulated by VE-822, inhibits the progression of lung adenocarcinoma in vitro and in vivo by deubiquitinating and stabilizing FHL1 (15,16), suggesting new potential targets for treating non-small cell lung cancer and lung adenocarcinoma. In recent years, increasing evidence has demonstrated that OTUD1 inhibits the NF-κB pathway, thereby negatively regulating inflammation and serving an important role in inflammatory diseases (17,18). Studies have shown that OTUD1 negatively regulates inflammatory responses by inhibiting the activation of transforming growth factor-β-activated kinase 1 (TAK1)-mediated MAPK and NF-κB signaling pathways, providing protection against sepsis-induced lung injury, which may be related to the ability of OTUD1 to deubiquitinate TIPE2 (14,19). Furthermore, OTUD1 specifically reverses K63-linked ubiquitination of RIPK1 and inhibits NEMO recruitment, suppressing RIPK1-mediated NF-κB activation and preventing intestinal inflammation (17). Studies have also shown that RIP2 mediates ischemic brain injury by promoting inflammatory responses, whereas OTUD1 improves post-ischemic brain injury by specifically cleaving K63 ubiquitination of RIP2 and inhibiting RIP2-induced NF-κB activation (18,20).
In the present study, bioinformatics analysis predicted the presence of N6-methyladenosine (m6A) methylation sites in the 3′UTR of OTUD1. Related literature has indicated that methyltransferase-like 3 (METTL3) can promote m6A methylation (21) and COPD progression, whereas METTL3 silencing can reduce OTUD1 mRNA m6A methylation, thereby enhancing OTUD1 protein expression (22). YTH m6A RNA binding protein 2 (YTHDF2), a member of the YT521-B homology domain family 2, is an m6A ‘reader’ that recognizes m6A modifications and triggers a series of downstream biological responses. Increasing evidence has indicated that YTHDF2 serves notable roles in various mechanisms across both cancerous and non-cancerous conditions in patients (23–25). Therefore, the present study aimed to investigate the role of OTUD1 in COPD pathogenesis and its regulation by METTL3-mediated m6A methylation. Specifically, the study aimed to characterize OTUD1 expression patterns in COPD progression using public datasets (GSE38974 and GSE69818) and cigarette smoke extract (CSE)-exposed BEAS-2B cells; elucidate the functional impact of OTUD1 on inflammation and pyroptosis through overexpression and knockdown models, with a focus on NF-κB signaling and inflammasome-related pathways; and delineate the mechanism by which METTL3-driven m6A methylation, in collaboration with the reader YTHDF2, suppresses OTUD1 expression under CSE exposure. The findings aim to uncover novel therapeutic targets for COPD by linking epigenetic regulation of OTUD1 to inflammatory dysregulation in smoke-induced lung injury.
Materials and methods
Cell culture
The normal human lung epithelial cell line, BEAS-2B, was obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. BEAS-2B cells were cultured in a cell incubator with 5% CO2 at 37°C using Dulbecco's Modified Eagle's medium (DMEM; cat. no. SH30243.01; Hyclone; Cytiva) supplemented with 10% fetal bovine serum (cat. no. 16000e044; Gibco; Thermo Fisher Scientific, Inc.). Additionally, to avoid bacterial contamination, 1% penicillin with streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) was added to the DMEM and the medium was replaced every 2–3 days.
Cell transfection
Cells were transfected with plasmids using Lipofectamine™ 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, cells were seeded at a density of 2×105 cells/well in 6-well plates 24 h prior to transfection to reach 70–80% confluence at the time of transfection. The concentrations of small interfering (si)RNAs and the pcDNA3.1 plasmid (Invitrogen; Thermo Fisher Scientific, Inc.) expressing OTUD1 (NM_001145373.3, CDS: 384–1,892) used for transfection were 50 nM and 2 µg per well, respectively. The empty pcDNA3.1 vector was used as a negative control for OTUD1 overexpression (vector group). The siRNAs were synthesized by Shanghai GenePharma Co., Ltd. Transfection was performed at 37°C in a humidified atmosphere with 5% CO2. Following transfection for 12 h, the cells were incubated for 48 h before being subjected to subsequent experiments. The sequences were as follows: OTUD1 siRNA (siOTUD1)-1 sense, 5′-AUCAUAGUGUCCGUUACUGAG-3′, antisense, 5′-CUCAGUAACGGACACUAUGAU-3′; siOTUD1-2 sense, 5′-UUUUAGACAAGGAUAUGGCCA-3′, antisense, 5′-UGGCCAUAUCCUUGUCUAAAA-3′; siOTUD1-3 sense, 5′-UUUGAAAGUGCUAUUACCCUG-3′, antisense, 5′-CAGGGUAAUAGCACUUUCAAA-3′; METTL3 siRNA (siMETTL3)-1 sense, 5′-UCUAACUCAGGAUCUGUAGCU-3′, antisense, 5′-AGCUACAGAUCCUGAGUUAGA-3′; siMETTL3-2 sense, 5′-UGUGUUUAUUGAUAAUUCGUC-3′, antisense 5′-GACGAAUUAUCAAUAAACACA-3′; siYTHDF2-1 sense, 5′-UUAAUCCAUCCUUUUGAUGUA-3′, antisense, 5′-UACAUCAAAAGGAUGGAUUAA-3′; siYTHDF2-2 sense, 5′-ACUUUUGGAACAUUGCUUGCA-3′, antisense, 5′-UGCAAGCAAUGUUCCAAAAGU-3′; siRNA-negative control (siNC) sense, 5′-UUCUCAGAACGUGUCACAU-3′, antisense: 5′-AUGUGACACGUUCUGAGAA-3′.
Preparation of CSE and cell treatment
The aim of the present study was to simulate the microenvironment of cigarette smoke exposure in cell culture. The CSE solution was prepared on the day of the experiment. First, commercial cigarettes (each containing 2.5 mg nicotine and 12 mg tar; purchased from Shanghai, China) were drawn into a flask containing 10 ml DMEM at a rate of 1 cigarette every 3 min using a vacuum pump. The pH of the CSE solution was adjusted to 7.4, and the OD (A320-A540) was maintained between 0.9 and 1.2. The CSE solution was then filtered through a 0.22-µm filter to remove bacteria and other microorganisms. This solution was considered to be 100% CSE and was further diluted with DMEM to a working concentration of 10% for subsequent experiments, which were performed within 1 h of preparation as needed. After transfection with siRNA, cells were treated with the prepared CSE solution to simulate the cell culture environment. The treatment was performed at 37°C for 24 h. Pyrrolidine dithiocarbamate (PDTC; 10 µM; cat. no. 52202ES50; Shanghai Yeasen Biotechnology Co., Ltd.), an NF-κB inhibitor that inhibits IκB phosphorylation, blocks NF-κB translocation into the nucleus and reduces the expression of downstream cytokines (26), was used to treat siOTUD1-transfected human BEAS-2B cells at 1 h post-transfection for 24 h at 37°C.
ELISA
Secreted IL-1β and IL-18 levels in the supernatant were determined by ELISA. The cultured cells were centrifuged at 1,000 × g for 20 min at 4°C, and after removing the cell pellets, the secreted IL-1β and IL-18 levels in the supernatant were quantified using human IL-1β and IL-18 ELISA kits (cat. nos. EK101B and EK118; Hangzhou Lianke Biology Technology Co., Ltd.), according to the manufacturer's instructions. The concentration of IL-1β and IL-18 was detected at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).
Pyroptosis assay
To detect cell pyroptosis, cultured cells (~1×106 cells) were resuspended in PBS. Subsequently, anti-active caspase-1 (1:1,000; cat. no. MA5-32137; Invitrogen; Thermo Fisher Scientific, Inc.) was added and incubated for 1 h at 25°C. The cells were then washed with PBS three times to remove non-combined caspase-1, followed by incubation with 3 µM propidium iodide solution (cat. no. P3566; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 15 min in the dark. Cell pyroptosis was assessed by flow cytometry (NovoCyte D3000; Agilent Technologies, Inc.) and analyzed using FlowJo 10.8.1 software (BD Biosciences), and the rate of cell pyroptosis was calculated.
Reporter gene assay
The OTUD1 3′UTR sequence was cloned into the pGL3 vector (Promega Corporation). BEAS-2B cells were co-transfected with siNC, siMETTL3-1 or siMETTL3-2 and the pGL3-OTUD1 3′UTR luciferase reporter plasmid, using Lipofectamine 3000 as the transfection reagent. The cells were cultured in 5% CO2 at 37°C for 24 h. Subsequently, the cells were lysed with lysis buffer (cat. no. 89901; Invitrogen; Thermo Fisher Scientific, Inc.), and the luciferase activity was measured by adding the Renilla-Firefly Luciferase Dual Assay Kit (MedChemExpress) luciferase substrate to the cell lysates. The luminescence was detected using a luminometer, and the luciferase activity was normalized to Renilla luciferase activity to assess the effects of METTL3 knockdown on OTUD1 3′UTR-mediated reporter expression.
RNA immunoprecipitation (RIP) and methylated RNA immunoprecipitation (meRIP) assay
The RIP assay was performed using the Magna MeRIP™ m6A Kit (MilliporeSigma). To isolate RNA-protein complexes, cells were cultured until they reached 70–80% confluence. RNA-protein complexes were crosslinked by adding 1% formaldehyde for 10–15 min at room temperature, and then quenched with 125 mM glycine for 5–10 min at 4°C and rinsed with cold PBS. The cells (1×107) were lysed with a lysis buffer containing protease, RNase and phosphatase inhibitors, and the cells were homogenized, as required. Then, the RNA-protein complexes were conjugated with antibodies (5 µg): Anti-YTHDF2 (cat. no. ab220163; Abcam) or anti-IgG (cat. no. ab172730; Abcam) at 4°C for 1 h. After which, the agarose beads and 50 µl protein A/G were added and incubated at 4°C for 1 h. The beads were subsequently rinsed with lysis buffer to remove unbound material, and RNA was extracted using TRIzol® (cat. no. 15596026CN; Invitrogen; Thermo Fisher Scientific, Inc.) and treated with DNase to remove DNA contamination. Finally, RNA was analyzed by reverse transcription-quantitative PCR (RT-qPCR).
For meRIP, the aforementioned RNA-protein complexes were conjugated with 5 µg anti-m6A antibody (cat. no. ab208577; Abcam) at 4°C for 1 h. The other processes were the same as those performed for the RIP experiment.
Actinomycin D treatment
A stock solution of actinomycin D (1 mg/ml in DMSO; MedChemExpress) was prepared. The stock solution was diluted to the desired working concentration (5 µg/ml) in culture medium. For treatment, the diluted actinomycin D solution was added to the culture medium at the desired concentration. It was ensured that the final concentration of DMSO did not exceed 0.1%. Cells were treated with actinomycin D at 37°C for 4 h. The treatment was applied after transfection to ensure that any potential effects of actinomycin D on transcription could be accurately assessed in the context of the transfected cells.
RT-qPCR
To quantify mRNA expression levels, RT-qPCR was performed. Briefly, total RNA was extracted using TRIzol reagent and quantified using a spectrophotometer. RNA was reverse-transcribed using the PrimeScript™ RT reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. The RT reaction was carried out using 1 µg RNA in a total volume of 20 µl, with the following reaction conditions: 37°C for 15 min, followed by 85°C for 5 sec to deactivate the reverse transcriptase enzyme. qPCR was carried out using SYBR Green qPCR Master Mix (Thermo Fisher Scientific, Inc.) on a C1000 thermal cycler (Bio-Rad Laboratories, Inc.) with the following cycling conditions: Initial denaturation at 95°C for 5 min; 40 cycles at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec; and a final extension step at 72°C for 5 min to ensure complete amplification. The primers used in the present study were self-designed based on the target gene sequences retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Primer design was carried out using Primer3 (version 0.4.0) (https://bioinfo.ut.ee/primer3-0.4.0/) to ensure appropriate melting temperatures and specificity. Primer sequences were synthesized by Sangon Biotech Co., Ltd. The Cq value was defined as the number of cycles needed for the fluorescent signal in the reaction tube to reach the set threshold. The relative mRNA levels normalized to GAPDH were calculated using the 2−ΔΔCq method (27). The primer sequences for RT-qPCR were designed as follows: GAPDH forward (F), 5′-CACCATCTTCCAGGAGCGAG-3′, reverse (R), 5′-TGATGACCCTTTTGGCTCCC-3′; OTUD1 F, 5′-TTTGGCTCAGTTGGCTCAGT-3′, R, 5′-CGCGTTTCCTTTGCACTTGA-3′; OTUD1 3′UTR F, 5′-CUUACACCCUGGGAAUAAUUG-3′, R, 5′-GATTAAGGCATTACACCTAC-3′; METTL3 F, 5′-GTGATCGTAGCTGAGGTTCGT-3′, R 5′-GGGTTGCACATTGTGTGGTC-3′; YTHDF2, F, 5′-CAGGCAAGGCCCAATAATGC-3′, R, 5′-AAGTAGGGCATGGCTGTGTC-3′.
Western blotting
The total protein was extracted from BEAS-2B cells using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.), and the BCA protein assay kit (cat. no. A55864; Thermo Fisher Scientific, Inc.) was used to determine the protein levels. Subsequently, the samples were boiled at 95°C for 10 min and proteins (20–40 µg/lane) were separated by SDS-PAGE on 10% gels at a constant voltage (80 V) until the dye front reached the bottom of the gel. The separated proteins were then transferred to a 0.2-µm PVDF membrane. For transfer, a constant current of 400 mA was applied for 1 h at 4°C to ensure efficient transfer of proteins. The membranes were then blocked with 5% non-fat milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.4) for 1 h at room temperature to prevent non-specific binding of antibodies, followed by incubation with the following primary antibodies: NF-κB p65 (1:1,000; cat. no. ab32536; Abcam), NF-κB phosphorylated (p-)p65 (1:1,000; cat. no. ab76302; Abcam), active caspase-1 p20 (1:1,000; cat. no. abs154965; Absin Bioscience, Inc.), gasdermin D N-terminal domain (GSDMD-N; 1:1,000; cat. no. ab215203; Abcam), NLR family pyrin domain containing 3 (NLRP3; 1:1,000; cat. no. ab283819; Abcam), METTL3 (1:1,000; cat. no. ab195352; Abcam), YTHDF2 (1:1,000; cat. no. ab220163; Abcam), OTUD1 (1:1,000; cat. no. 29921-1-AP; Proteintech Group, Inc.), and GAPDH (1:5,000; cat. no. 60004-1-Ig; Proteintech Group, Inc.) at 4°C overnight with gentle agitation. The membrane was washed three times with TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000; cat. nos. A0208 and A0216; Beyotime Institute of Biotechnology) diluted in TBST containing 2% BSA (cat. no. 9048-46-8; Absin Bioscience, Inc.) for 1 h at 25°C. Finally, the expression levels of proteins were measured using a chemiluminescent imaging system (Tanon 5200; Tanon Science and Technology Co., Ltd.).
Methylation site prediction
The present study employed a bioinformatics pipeline to predict methylation sites in the OTUD1 gene. The full-length mRNA sequence of Homo sapiens OTUD1 (accession no. NM_001145373.3, including 5′-UTR, CDS and 3′-UTR regions) was retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/nuccore/NM_001145373.3). CpG island and methylation region analyses were performed using the MethPrimer platform (https://www.urogene.org/methprimer/), which integrates sequence features (CpG dinucleotide density and GC content; thresholds: Length >200 bp, GC content >50%, observed/expected CpG ratio >0.6) with a machine learning model (random forest algorithm) to identify potential methylation sites, supplemented by cross-species conservation annotations. The ‘full prediction’ mode was selected for whole-sequence scanning, with RNA secondary structure analysis disabled, and a confidence threshold >0.7. Bioinformatics analysis using the SRAMP prediction server predicted the presence of an m6A methylation modification site in the OTUD1 3′UTR (http://www.cuilab.cn/m6asiteapp/old), and the predicted sites were validated against conserved regions in the MethDB (http://www.methdb.net/) and MethBank (https://ngdc.cncb.ac.cn/methbank/) databases, and CpG island-gene domain correlations were visualized via the UCSC Genome Browser (https://genome.ucsc.edu/).
Statistical analysis
The relevant data from two datasets in the Gene Expression Omnibus database [GSE38974 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38974) and GSE69818 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69818)] were integrated for subsequent analyses of differences in gene expression. Each experiment was performed in triplicate and data are presented as the mean ± standard deviation of three independent biological replicates. Statistical analyses were conducted using GraphPad Prism 7.0 software (Dotmatics). To compare differences in mean values among multiple groups, one-way analysis of variance followed by Tukey's post hoc test was applied. For comparisons between two groups, an unpaired two-tailed Student's t-test was used. P<0.05 was considered to indicate a statistically significant difference.
Results
Cigarette smoke stimulation inhibits OTUD1 expression
Analysis of COPD datasets GSE38974 (28) and GSE69818 (29) revealed that the expression levels of the OTUD1 gene were lower in advanced COPD compared with those in early COPD (Fig. S1). Subsequently, OTUD1 expression was detected in BEAS-2B cells treated with 10% CSE for 0, 12, 24 and 48 h using western blotting and RT-qPCR. RT-qPCR and western blotting results indicated that 10% CSE stimulation inhibited the expression levels of OTUD1 (Fig. 1A-C), demonstrating that cigarette smoke stimulation can decrease OTUD1 gene expression in human BEAS-2B cells.
OTUD1 reduces cellular damage caused by cigarette smoke stimulation by inhibiting the inflammatory response
To further verify that OTUD1 has a protective effect on cells stimulated with 10% CSE, an OTUD1 overexpression (oeOTUD1) plasmid was constructed and transfected into human BEAS-2B cells, after which, RT-qPCR and western blotting were used to verify the transfection efficiency of oeOTUD1 (Fig. 1D-F). Subsequently, human BEAS-2B cells were treated with 10% CSE, and pyroptosis was detected by flow cytometry (Fig. 2A and B). The results showed that pyroptosis was significantly increased in the 10% CSE-treated group compared with that in the vector group, whereas pyroptosis was reduced in the 10% CSE-treated oeOTUD1 group compared with that in the group treated with 10% CSE alone. To investigate whether the protective effect of OTUD1 on human BEAS-2B cells was related to inflammation, the levels of pro-inflammatory factors, NF-κB pathway proteins (p65 and p-p65) and proteins related to inflammasomes (NLRP3, p20 and GSDMD-N) were further detected. Common pro-inflammatory cytokines IL-1β and IL-18 were selected, and the levels of IL-1β and IL-18 in cell supernatants were detected by ELISA. The results showed that 10% CSE stimulation of human BEAS-2B cells resulted in a significant increase in IL-1β and IL-18 compared with that in normal human BEAS-2B cells; however, overexpression of OTUD1 could partially inhibit the elevation of IL-1β and IL-18 induced by cigarette smoke (Fig. 2C and D). To some extent, these findings verified that OTUD1 could serve a protective role in cells by reducing inflammation under cigarette smoke stimulation conditions. To further verify this hypothesis, NF-κB p65/NF-κB p-p65, NLRP3, active caspase-1 p20 and GSDMD-N were detected by western blotting. The results showed that the expression levels of NF-κB p65/NF-κB p-p65, NLRP3, active caspase-1,p20 and GSDMD-N were significantly increased in the cell group treated with 10% CSE, whereas they were reduced in the oeOTUD1 group (Fig. 2E-I). These findings further validated that OTUD1 expression may serve a protective role in cells by inhibiting inflammation.
Reverse validation of the protective effect of OTUD1 on cells through inhibition of the inflammatory response in BEAS-2B cells
In the present study, reverse validation was performed, whereby siOTUD1-transfected human BEAS-2B cells were constructed, and the transfection efficiency of siOTUD1 was verified by RT-qPCR and western blotting (Fig. 3A-C). The cells were divided into the following three siRNA groups: i) siOTUD1-1; ii) siOTUD1-2; and iii) siOTUD1-3. And the siOTUD1-1was used in subsequent experiments, as it induced lower OTUD1 protein levels. The results of the pyroptosis assay suggested that pyroptosis was significantly increased after knockdown of OTUD1, whereas the addition of the NF-κB inhibitor PDTC resulted in a decrease in pyroptosis compared with that in the siOTUD1 group (Fig. 3D and E). Subsequently, ELISA was performed to detect the levels of IL-1β and IL-18 in cell supernatants; the results were consistent with the pyroptosis assay results, after silencing the expression of OTUD1, the levels of the pro-inflammatory cytokines IL-1β and IL-18 were significantly increased, whereas this increase was inhibited by the addition of the NF-κB inhibitor PDTC (Fig. 3F and G). Furthermore, the expression levels of NF-κB p65/NF-κB p-p65, NLRP3, active caspase-1 p20 and GSDMD-N were examined by western blotting, and the results also showed that the expression of these proteins were markedly increased after silencing the expression of OTUD1, whereas the NF-κB inhibitor PDTC could attenuate the elevated inflammatory response induced by OTUD1 knockdown (Fig. 3H-L). These findings indicated that OTUD1 could serve a protective role in patients with COPD and that this protective effect may be achieved through its anti-inflammatory effects.
CSE stimulation promotes OTUD1 methylation by increasing METTL3 activity, which in turn inhibits OTUD1 gene expression
Relevant studies have shown that METTL3 expression is significantly elevated in patients with smoking-induced COPD and COPD cell models, and that METTL3-mediated modification of m6A RNA methylation regulates CSE-induced EMT by targeting OTUD1 mRNA, ultimately serving a key regulatory role in the emergence of COPD (30–32). Bioinformatics analysis predicted the presence of an m6A methylation modification site in the OTUD1 3′UTR. meRIP-qPCR of OTUD1 methylation levels in human BEAS-2B cells treated with 10% CSE demonstrated that the m6A methylation level was significantly elevated in the 10% CSE-treated cells compared with that in the control group (Fig. 4A), which indicated that 10% CSE stimulation could inhibit OTUD1 expression by increasing m6A.
After human BEAS-2B cells were treated with 10% CSE, METTL3 expression was detected by RT-qPCR and western blotting; the results showed that the expression levels of METTL3 were elevated in the 10% CSE group (Fig. 4B-D). These findings indicated that cigarette smoke stimulation may increase m6A methylation through increasing METTL3 activity levels, which in turn inhibits the expression of OTUD1.
To provide further verification, 10% CSE-treated human BEAS-2B cells transfected with siMETTL3 underwent meRIP-qPCR to detect the m6A methylation level of OTUD1. RT-qPCR and western blotting were used to detect the expression levels of OTUD1, and a luciferase reporter gene was used to detect the 3′UTR activity of OTUD1. The results showed that the methylation level of OTUD1 was decreased, and the expression levels of OTUD1 and the 3′UTR activity of OTUD1 were increased in cells transfected with siMETTL3 and stimulated with 10% CSE compared with those in the group stimulated with 10% CSE alone (Fig. 4E-L). Therefore, by knocking down METTL3, it was further verified that 10% CSE stimulation may promote OTUD1 methylation by increasing METTL3 activity, which in turn suppresses OTUD1 gene expression. Since transfection with siMETTL3-1 resulted in the lowest METTL3 protein levels, siMETTL3-1 was used in the subsequent experiments.
Inhibition of the m6A methylation reader YTHDF2 mitigates the increased methylation of OTUD1 induced by 10% CSE
To assess the transfection efficiency of siYTHDF2, the expression levels of YTHDF2 were detected in human BEAS-2B cells transfected with siYTHDF2 using RT-qPCR and western blotting (Fig. 5A-C). siYTHDF2-1 was used in the subsequent experiments as it induced lower YTHDF2 protein levels than siYTHDF2-2. Actinomycin D, a transcription inhibitor, forms stable complexes by intercalating into DNA, blocking DNA-dependent RNA polymerase activity, and thereby inhibiting transcription, making it a common tool for RNA stability assays (16). BEAS-2B cells with YTHDF2 knockdown were treated with 10% CSE and 5 µg/ml actinomycin D for 0.5, 3 and 6 h, followed by RT-qPCR to assess OTUD1 transcription levels. The results showed that OTUD1 expression was higher in the group treated with 10% CSE combined with YTHDF2 knockdown compared with that in the group treated with 10% CSE alone (Fig. 5D), thus indicating that YTHDF2 inhibition also suppressed m6A methylation, thereby partially relieving the suppression of OTUD1 expression.
To validate this hypothesis, BEAS-2B cells were treated with 10% CSE, and meRIP-qPCR was performed to detect the binding of YTHDF2 to the 3′UTR of OTUD1. The results indicated that cigarette smoke exposure increased the binding of YTHDF2 to the OTUD1 3′UTR (Fig. 5E). Furthermore, in BEAS-2B cells treated with 10% CSE alone or combined with METTL3 knockdown, meRIP-qPCR was used to assess YTHDF2 binding to the OTUD1 3′UTR. The results showed that METTL3 inhibition reduced the binding of YTHDF2 to the OTUD1 3′UTR (Fig. 5F).
Discussion
Recent studies have shown that OTUD1 regulates the NF-κB pathway primarily by inhibition, inducing protective effects on sepsis-induced lung injury, inflammatory bowel disease and inflammation-mediated ischemic brain injury (17,18). Specifically, OTUD1 inhibits the activation of certain inflammatory pathways, such as the NF-κB pathway, by removing ubiquitin from key signaling proteins (33). This deubiquitination activity prevents the activation of NF-κB, which serves a central role in promoting inflammation. For example, in the NF-κB pathway, OTUD1 serves a role in preventing the activation of NF-κB by removing K63-linked ubiquitin chains from RIPK1. This de-ubiquitination process reduces the ability of NEMO (a key protein in the NF-κB signaling pathway) to bind to RIPK1, thus suppressing the activation of NF-κB signaling. Essentially, OTUD1 acts as a negative regulator of this pathway (34). In addition, OTUD1 inhibits TAK1-mediated MAPK and NF-κB pathways (35) to reduce inflammation in conditions such as sepsis-induced lung injury and ischemic brain injury. Furthermore, OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory and oxidative stress responses (34). Overall, OTUD1 acts as a negative regulator of these pathways by inhibiting their activation, which aids suppression of excessive inflammation. The anti-inflammatory effects of OTUD1 may indicate a novel role in COPD.
In the present study, the analysis of COPD datasets (GSE38974 and GSE69818) revealed that OTUD1 expression was lower in advanced COPD compared with that in the earlier stage. To assess the role of OTUD1 in COPD progression, BEAS-2B cells were stimulated with 10% CSE, and the results indicated that OTUD1 expression was decreased under CSE. To further confirm the role of OTUD1, oeOTUD1 models were constructed in BEAS-2B cells. Under similar CSE conditions, cells in the oeOTUD1 group exhibited reduced pyroptosis, decreased levels of the pro-inflammatory cytokines IL-1β and IL-18, and reduced expression levels of NF-κB and inflammasome-related proteins compared with those in the control group. These findings suggested that OTUD1 may reduce cell damage and pyroptosis induced by 10% CSE through inhibiting inflammation. To validate this further, siOTUD1 models were constructed and cells were also treated with the NF-κB inhibitor PDTC. The results showed that siOTUD1 aggravated cell pyroptosis, increased the levels of the pro-inflammatory cytokines IL-1β and IL-18, and enhanced the expression levels of NF-κB and inflammasome-related proteins. However, the addition of PDTC, an NF-κB inhibitor, reduced the levels of pyroptosis and inflammation. These findings further confirm that OTUD1 may reduce cell damage induced by CSE by inhibiting inflammatory responses.
METTL3 and m6A are frequently dysregulated in various pathological processes, controlling the expression of specific genes and regulating cellular functions (36–38). Additionally, METTL3 and m6A are actively involved in the pathogenesis of pulmonary diseases, including chronic conditions such as pulmonary fibrosis, pulmonary arterial hypertension and COPD, as well as acute diseases such as pneumonia, SARS-CoV-2 infection and sepsis-induced acute respiratory distress syndrome (39,40). The RNA m6A modification mediated by METTL3 represents a novel mechanism underlying the onset and progression of these pulmonary diseases. Related research has indicated that METTL3 expression is notably increased in patients with smoking-induced COPD and COPD cell models (41,42). Furthermore, METTL3 silencing has been reported to inhibit the EMT process induced by CSE in human bronchial epithelial cells (BEAS-2B) (22). Mechanistically, METTL3 silencing reduced m6A methylation of OTUD1 mRNA in the current study, thereby enhancing OTUD1 protein expression. Bioinformatics analysis predicted the presence of m6A methylation sites in the 3′UTR of OTUD1. To explore the association between METTL3-mediated m6A methylation and OTUD1 expression, BEAS-2B cells were treated with 10% CSE, and RT-qPCR and western blotting were used to detect METTL3 expression. The results showed that METTL3 levels were increased following 10% CSE stimulation, leading to elevated m6A methylation of OTUD1 and reduced OTUD1 expression. Upon constructing siMETTL3 models, m6A methylation levels were decreased whereas OTUD1 expression was increased, suggesting that 10% CSE stimulation promotes m6A methylation via increased METTL3 expression, thereby inhibiting OTUD1 expression. Subsequently, interference models were constructed for the m6A methylation reader YTHDF2. Following cigarette smoke exposure, siYTHDF2 knockdown alleviated the decrease in OTUD1 expression caused by cigarette smoke. These findings suggested that cigarette smoke exposure may inhibit OTUD1 expression by promoting m6A methylation, a process that requires the catalytic activity of METTL3 and the involvement of the reader YTHDF2. Cigarette smoke also increased the binding of YTHDF2 to the 3′UTR of OTUD1, which was partially suppressed by METTL3 knockdown. In conclusion, cigarette smoke exposure could increase the m6A methylation of OTUD1 involving METTL3 and YTHDF2, thereby inhibiting OTUD1 expression.
While previous studies have suggested that OTUD1 has tumor-suppressive properties, often linked to its ability to increase apoptosis in cancer cells (43,44), the findings of the present study indicated that OTUD1 may reduce pyroptosis in the context of COPD. This apparent contradiction may arise from the differing biological contexts in which OTUD1 functions. In cancer, the inhibition of NF-κB and other inflammatory pathways by OTUD1 often results in increased apoptosis of tumor cells, which is beneficial for limiting tumor progression (45). However, in COPD, the role of OTUD1 may shift towards protecting healthy lung cells from excessive inflammatory responses, such as pyroptosis, which is commonly induced by cigarette smoke exposure (46).
In the present study, oeOTUD1 reduced pyroptosis in BEAS-2B cells treated with CSE, suggesting that OTUD1 serves a protective role in the lungs under inflammatory stress conditions. This protective effect is likely due to its ability to inhibit excessive inflammation by suppressing NF-κB activation and inflammasome-related proteins, such as NLRP3 and GSDMD-N. Through this mechanism, OTUD1 may reduce cell damage and maintain cellular integrity, which contrasts with its pro-apoptotic effect in the context of cancer cells, where inflammation and immune evasion are significant factors. Further investigation into the specific mechanisms by which OTUD1 modulates inflammation and pyroptosis in different disease contexts will help to clarify its dual role in cellular responses.
In conclusion, in patients with COPD affected by cigarette smoke exposure, OTUD1 expression may decrease as the disease progresses. The OTUD1 gene reduces cell damage and inflammation caused by cigarette smoke exposure by inhibiting inflammatory responses. CSE (10%) was shown to inhibit OTUD1 expression by upregulating METTL3, which may enhance METTL3-mediated methylation and promote YTHDF2 recognition of OTUD1 methylation. Although the present study revealed the potential role of OTUD1 through database analysis and cellular experiments, these findings have not yet been validated in patient samples. Future research should use clinical data to analyze OTUD1 expression in tissue samples obtained from patients with COPD and to assess its association with disease severity, with the aim of enhancing the clinical applicability of these findings.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
The present study was supported by the Natural Science Foundation of China (grant no. 2023YFC3043507), Shanghai Pudong Hospital and the Discipline Construction Promoting Project of Shanghai Pudong Hospital (grant no. Zdzk2020-11).
Availability of data and materials
The data generated in the present study may be requested from the corresponding author.
Authors' contributions
JG, ZS, WT, ZW and YS designed the project. ZS, WT, JX, WC and ZC performed experiments, data analysis and interpretation. The manuscript was drafted by JG and YS. Study supervision was carried out by ZW and YS. ZW and YS confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
COPD |
chronic obstructive pulmonary disease |
OTUD1 |
ovarian tumor protease domain-containing protein 1 |
CSE |
cigarette smoke extract |
m6A |
N6-methyladenosine |
METTL3 |
methyltransferase-like 3 |
YTHDF2 |
YTH m6A RNA binding protein 2 |
RT-qPCR |
reverse transcription-quantitative PCR |
RIP |
RNA immunoprecipitation |
TAK1 |
transforming growth factor β-activated kinase 1 |
EMT |
epithelial-mesenchymal transition |
GSDMD-N |
gasdermin D N-terminal domain |
NLRP3 |
NLR family pyrin domain containing 3 |
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