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METTL14 modulates the progression and ferroptosis of colitis by regulating the stability of m6A-modified GPX4

European Journal of Medical Research volume 30, Article number: 88 (2025) Cite this article

Abstract

Ulcerative colitis (UC) is non-specific inflammatory bowel disease. UC development and progression were closely associated with epigenetic modifications. Nevertheless, the specific relationship between N6-methyladenosine (m6A) modification at RNA transcription levels and UC pathogenesis remains unclear. We established UC cell models and mouse models through dextran sulfate sodium (DSS) induction. The expression levels of METTL14 were analyzed via qRT-PCR and western blot. In vitro functional experiments evaluated the effects of METTL14 overexpression on the viability of DSS-induced NCM460 cells and ferroptosis markers. Use of the m6A methylation detection kit, MeRIP-qPCR, and RNA stability experiments confirmed the molecular mechanism controlled by METTL14. In vivo experiments with inflammatory mice models elucidated the interaction between METTL14 and GPX4. Findings from this study indicated a notable reduction in m6A methyltransferase METTL14 expression in DSS-induced NCM460 cells and DSS-induced mice models. METTL14 overexpression effectively suppressed ferroptosis in DSS-induced NCM460 cells. In addition, METTL14 enhanced GPX4 mRNA stability through mediating m6A modification, and the interplay between METTL14 and GPX4 through m6A modification introduced innovative therapeutic approaches for UC management.

Introduction

Inflammatory bowel disease (IBD) encompasses Crohn’s disease (CD) and ulcerative colitis (UC) [1]. UC, a chronic and non-specific intestinal condition, is characterized by recurrent manifestations such as abdominal pain, diarrhea, mucus in stools, and rectal bleeding [2]. The disease course is protracted and challenging to treat, with a noteworthy propensity for UC to progress to cancer [3]. While the exact cause of UC remains unclear, research indicates that genetic factors, intestinal microbiota, environmental influences, and immune system dysregulation may contribute to its pathogenesis [4,5,6]. The complexity of UC pathogenesis coupled with the lack of definitive cures has intensified research efforts in this area.

Advances in investigating the genetic underpinnings of UC have revealed an association between methylation modifications and the development of UC [7]. Among the prevalent internal modifications, N6-methyladenosine (m6A) stands out [8]. This dynamic and reversible m6A modification process is regulated by methyltransferases and demethylases [9]. Two key members of the methyltransferase group, METTL3 and METTL14, play significant roles in influencing the m6A methylation process and consequently impacting the host’s pathological progression [10, 11]. Research has shown that mesenchymal stem cells promoted the secretion of exosomal miR-34a-5p to enhance intestinal barrier function through METTL3/IGF2BP3-mediated Pre-miR-34A m6A modification [12]. METTL14 deficiency in m6A methyltransferase in T cells led to spontaneous colitis in mice, which was associated with Tregs dysfunction [13]. METTL14-mediated m6A modification was involved in various inflammatory diseases onset and progression. However, the specific molecular mechanisms by which METTL14 participates in the occurrence and progression of UC through m6A modification are not yet fully understood.

Ferroptosis, serving as a regulatory mechanism of cell death, is intricately linked to reactive oxygen species accumulation. Its primary characteristics encompass iron accumulation and lipid peroxidation [14, 15]. Recent research has unveiled a significant correlation between ferroptosis and inflammatory diseases progression [16]. Research studies have demonstrated that UC pathogenesis was associated with iron accumulation, lipid peroxidation accumulation, glutathione (GSH) depletion, GPX4 inactivation, and the enhancement of fatty acid oxidation enzymes [17]. Xu et al. discovered that ferroptosis-induced cell death was involved in the mechanism of intestinal epithelial cell death in UC [18]. Moreover, supplementing with GPX4 reducing agent, GSH, has the potential to notably enhance colonic health [19]. Dai et al. found that Nrf2-GPX4 signaling pathway was capable of substantially suppressing iron-induced cell death, but this pathway was suppressed in UC. Activating GPX4 can ameliorate UC symptoms [20]. Therefore, this study also emphasizes investigating the regulatory function of genes associated with ferroptosis.

This study delved into the impact of m6A methyltransferase METTL14 on UC pathogenesis, investigating its functional role and elucidating the mechanism of METTL14-mediated m6A modification in UC progression. The objective is to establish a novel theoretical foundation for comprehending the pathogenesis and targeted therapeutic approaches for this disease.

Materials and methods

Cell culture

The normal human colon mucosal epithelial cell line NCM460 was obtained from the American Type Culture Collection (ATCC, USA). The cells were cultured in the RPMI1640 (A4192301, Invitrogen, Carlsbad, USA) complete medium supplemented with 10% FBS (A5256701, Invitrogen), 1% penicillin/streptomycin (15140122, Invitrogen). Cultures were maintained in a 37 °C incubator with 5% CO2.

To establish the cell inflammation model: NCM460 cells were initially cultured overnight in serum-free medium, then treated with 0.8 μg/mL Dextran Sulfate Sodium (DSS) (160110, MP Biomedicals, San Francisco, USA) for 12 h. The cells were cultured in the RPMI1640 complete medium supplemented with 10% FBS and 1% double antibiotics incubated at 37 °C. Subsequently, the cells were harvested for further experiments.

Cell transfection

Following the Lipofectamine™ 3000 (L3000001, Invitrogen) manual protocol, plasmids encoding METTL14 (OE-METTL14), blank plasmids (OE-NC), GPX4 inhibitor (sh-GPX4) or NC inhibitor (sh-NC) (Ribobio, Guangzhou, China) was individually transfected into the cells at a concentration of 50 nM each. The concentration refers to the study of Pan et al. [21]. The primer sequences are shown in Table 1. The transfection process was conducted for 48 h, after which the cells were collected. Subsequently, the collected cells were processed according to the previously described cell inflammation model to induce an in vitro inflammatory response. Untreated NCM460 cells were employed as the negative control group. Finally, all cell samples were gathered for analysis.

Table 1 Primer sequences used for cell transfection
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qRT-PCR

According to the experimental methods mentioned by Ji et al. [22], total RNA was extracted using TRIzol reagent (12183555CN, Invitrogen), reverse transcribed into cDNA using a reverse transcription kit (6210A, Takara Biotech, Otsu, Japan), and analyzed by real-time fluorescence quantitative PCR to evaluate the relative expression levels of specific genes in each sample. GAPDH mRNA served as the internal control for assessing METTL14 and GPX4 mRNA expression. The relative gene expression levels were calculated using the 2−ΔΔCT method. The primer sequences are as followed in Table 2.

Table 2 Primers used for qRT-PCR
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Western blot

This experiment was conducted with reference to previous studies [23]. NCM460 cells from various treatment groups were transfected with pre-chilled PBS (C0221A, Beyotime, Shanghai, China), washed, and subsequently lysed with RIPA lysis buffer (P0013B, Beyotime) on ice for 30 min. The lysed cells were then centrifuged at 12,000×g for 15 min at 4 °C. Subsequently, 30 μg of protein was extracted for SDS-PAGE gel electrophoresis. Protein samples of each group were fully transferred to a PVDF membrane (ISEQ00010, Millipore, Billerica, USA), which was blocked with 5% skimmed milk powder for 90 min. The membrane was incubated overnight at 4 °C with primary antibodies against METTL14 (ab309096, 1:1000, Abcam, Cambridge, UK), GPX4 (ab125066, 1:1000, Abcam, Cambridge, UK), and GAPDH (ab181602, 1:10000, Abcam, Cambridge, UK). This was followed by incubation with secondary antibodies (ab205718, 1:2000, Abcam, Cambridge, UK) for 1.5 h at 37 °C. After three TBST (T9039, Sigma-Aldrich, St. Louis, USA) washes, visualization was performed using ECL chemiluminescence, with GAPDH used as the internal reference. Finally, grayscale intensity analysis was performed using ImageJ software to quantify protein expression.

CCK-8 assays

NCM460 cells from different treatment groups were seeded in a 96-well plate at a density of 2,000 cells per well. Then, 10 μL CCK-8 (C0038, Beyotime) was added to each well, followed by incubation at 37 °C in a temperature-controlled incubator for 4 h. The absorbance at 450 nm was measured using an absorbance reader (BioTek, Winooski, VT, USA). The CCK-8 assay was conducted as previously described [24].

Reactive oxygen species (ROS) levels

The specific procedure was based on the research conducted by Chen et al. [25]. Inoculating NCM460 cells in the logarithmic growth phase into a 6-well plate (1000 cells/well), continuing cultivation for 24 h, add 10 μM DCFH-DA fluorescence probe (35845-5G, Sigma-Aldrich) to each well (excluding the blank group), and incubate in a light-avoiding cell culture incubator at 37 °C for 30 min. Wash the cells three times with PBS (C0221A, Beyotime), to thoroughly remove any residual DCFH-DA dye that has not entered the cells or remains in the solution. Finally, harvest cells from each group and measure the fluorescence intensity using a fluorescent microscope.

Glutathione (GSH), malondialdehyde (MDA) and iron levels

NCM460 cells from various treatment groups were seeded in a 6-well plate and cultured until reaching 60–70% confluence. Following two washes with PBS (C0221A, Beyotime), a minimal amount of PBS was added, and cells from each group were harvested. The levels of iron, GSH, and MDA were assessed using iron (#A039-2-1, Nanjing Jincheng, Nanjing, China), GSH (#A006-2-1, Nanjing Jincheng), and MDA (#A003-4-1, Nanjing Jincheng) assay kits, respectively. The specific procedure was carried out according to the instructions provided in the reagent manuals.

Total m6A RNA detection

Total RNA was isolated from NCM460 cells of diverse treatment groups and quantified the m6A levels in each group utilizing an m6A RNA methylation quantification kit (ab185912; Abcam). The specific procedure was carried out according to the instructions provided in the reagent manual.

MeRIP-qPCR

MeRIP-qPCR was performed using previously described methods [26]. Total RNA from each cell group was extracted using Trizol (12183555CN, Invitrogen), followed by purification with a PolyA mRNA purification kit. The m6A antibody (ab208577, Abcam) and IgG antibody (ab109489, Abcam) were individually introduced into the immunoprecipitation buffer and incubated with protein A/G magnetic beads for 1 h. Subsequently, the RNA and bead–antibody complexes were incubated overnight at 4 °C. The bound RNA was eluted using elution buffer, purified through phenol–chloroform extraction, and subjected to qPCR analysis.

RNA stability

The RNA stability was evaluated by measuring the RNA half-life as previously described [27]. NCM460 cells from various treatment groups were plated in a 6-well dish until they reached the logarithmic growth phase. Following this, 1 μg/mL actinomycin D (SBR00013, Sigma-Aldrich) was applied, and total RNA was extracted from the cells at intervals of 2, 4, 6, and 8 h to assess RNA stability through qRT-PCR.

Animal experiment

Eighteen male BALB/c mice aged 6 to 8 weeks were selected for the animal experiment, with the mouse experimental protocol approved by the hospital’s Institutional Animal Care and Use Committee. The mice were randomly divided into 6 groups of 3 mice each: control, DSS, DSS + oe-NC, DSS + oe-METTL14, DSS + oe-METTL14 + sh-NC, and DSS + oe-METTL14 + sh-GPX4. Mice in the DSS model group were administered a solution containing 3% DSS for 7 days to establish UC model in mice. In the control group, mice were provided with normal drinking water and did not undergo DSS modeling or any other special treatment. Transfect NCM460 cells with oe-NC, oe-METTL14, oe-METTL14 + sh-NC, and oe-METTL14 + sh-GPX4 at a viral titer of 1 × 107. After the model establishment, the mice were anesthetized with an intraperitoneal injection of pentobarbital sodium at a dosage of 1%. The transfected cells were then injected into the colonic region of the mice for three consecutive days, while both the DSS group and the normal group received an equal volume of PBS. Weight loss was recorded daily, and the DAI was scored as previously described. On the eighth day, the mice were euthanized by cervical dislocation after deep anesthesia, and the colon length was measured. 1–2 cm segment near the anus of the colon was selected for immunohistochemistry, Western blot analysis, and ELISA testing. All animal procedures were approved by the ethics committee of Dongguan Hospital of Traditional Chinese Medicine (MDKN-2023-051).

Disease activity index (DAI) score

For the colitis mouse model, we evaluated the modeling success using the DAI score [28], as shown in Table 3. This index assesses three aspects: weight, fecal consistency, and fecal occult blood. The DAI score was calculated as the average of these three indicators. DAI score exceeding 0.5 signified the successful establishment of the colitis mouse model.

Table 3 Scoring criteria for DAI
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ELISA

The colon tissues from each group of mice were harvested, homogenized in PBS (C0221A, Beyotime) using a homogenizer, centrifuged at 3000 rpm for 20 min, and the resulting supernatant was collected. The levels of pro-inflammatory factors, TNF-α and IL-1β, were determined utilizing ELISA kits (P0205M, Beyotime) as per the manufacturer’s instructions.

Hematoxylin and eosin (H&E) staining

Tissue histopathologically changed involves analyzing colon tissue stained with H&E under an optical microscope to assess colonic structural damage, inflammatory cell infiltration, and overall modeling efficacy. The H&E assays were conducted as previously described [29]. Fresh colon tissue was collected, longitudinally cut, and immersed in formalin (R04586, Sigma-Aldrich) overnight, paraffin-embedded, sectioned, deparaffinized, hydrated, and stained with H&E (G1076, Servicebio, Wuhan, China). Colonic inflammation cell infiltration and compromised intestinal epithelial mucosal barrier were then observed using an inverted microscope. Subsequently, tissue pathology scoring was conducted to compare the extent of intestinal mucosal inflammation among the groups [30]. The histological score of colitis comprised two components: the epithelial injury score and the immune cell infiltration score. (1) Epithelial injury score: normal = 0, cup shape cell loss = 1, goblet cell loss = 2, crypt loss = 3; (2) immune cell infiltration score: no infiltration = 0, infiltration around the crypt = 1, mucosal muscular infiltration = 2, submucosal infiltration = 3. The combined histological score ranges from 0 (no changes) to 6 (extensive cell infiltration and tissue damage).

Statistical analysis

Statistical analysis and data visualization were performed using GraphPad Prism 8.0 software. Each cell experiment group comprised 3 replicates. Measurement data were expressed as mean ± standard deviation (SD). A t-test was utilized for comparison between the two groups, while one-way analysis of variance (ANOVA) was employed for intergroup comparisons. p < 0.05 indicated statistically significant differences.

Results

METTL14 expression was downregulated in both cells and mice following treatment with DSS

After inducing NCM460 cells with DSS for 24 h, qRT-PCR was employed to quantify METTL14 expression levels in cell and mice models of IBD. The in vitro experiments showed a notable reduction in METTL14 mRNA and protein expression levels in the inflammatory cell model group, as opposed to the control group (Fig. 1A, B). This trend was similarly observed in the inflammatory bowel mice model groups (Fig. 1C, D), indicating that the METTL14 expression was in negatively correlated with the progression of inflammation.

Fig. 1
figure 1

METTL14 was downregulated in DSS-treated NCM460 cells and acute inflammatory bowel disease mice. A, B The mRNA and protein levels of METTL14 in NCM460 cells treated with DSS were assessed using qRT-PCR and western blot. C, D The mRNA and protein levels of METTL14 were measured in colon tissues of mice with acute inflammatory bowel disease induced by DSS treatment. ***p < 0.001

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METTL14 overexpression inhibited ferroptosis in DSS-treated NCM460 cells

The decreased expression of METTL14 in the aforementioned DSS-treated cells and mice models prompted the subsequent overexpression of METTL14 through cell transfection methods in our experiments to investigate its specific biological functions. Utilizing qPCR-PCR and Western blotting techniques, we successfully engineered NCM460 cells with overexpressed METTL14 (Fig. 2A, B). Subsequent modeling after DSS induction showed that the diminished cell viability caused by DSS was partly reversed by METTL14 overexpression (Fig. 2C). Notably, in our study, we observed a significant attenuation of elevated ROS levels induced by DSS modeling upon overexpressing METTL14 (Fig. 2D). Moreover, GPX4 suppression in DSS-induced NCM460 cells was mitigated by transfecting oe-METTL14 (Fig. 2E). GPX4, an essential selenium protein, facilitates the specific conversion of lipid hydroperoxides into lipid alcohols via GSH and plays a vital role in governing cellular ferroptosis [31]. Considering METTL14 regulatory impact on GPX4, our subsequent experiments focused on assessing the impact of METTL14 on ferroptosis progression. The findings revealed that the decrease in GSH levels and the increase in MDA and iron levels by DSS was restored by the overexpressing of METTL14 (Fig. 2F–H). The results showed that overexpression of METTL14 impeded ferroptosis in cells by increasing GPX4 expression but also by reducing lipid peroxidation and iron accumulation.

Fig. 2
figure 2

METTL14 overexpression inhibited ferroptosis in DSS-treated NCM460 cells. A, B Transfection efficiency of METTL14 was assessed using qRT-PCR and western blot. C Cell viability in DSS-treated NCM460 cells was determined by CCK-8. D ROS levels in NCM460 cells treated with DSS were assessed using the ROS assay kit. E GPX4 expression in NCM460 cells from various treatment groups was assessed using Western blot analysis. F–H The levels of GSH (F), MDA (G), and iron (H) in NCM460 cells from various treatment groups were assessed using the GSH, MDA, and iron assay kits. *p < 0.05, **p < 0.01, ***p < 0.001

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METTL14 improved the stability of m6 modification in GPX4 mRNA

Therefore, we investigated the impact of METTL14 on m6A levels in GPX4. Our experimental assay kit assessed the effect of DSS-induced modeling on m6A methylation in NCM460 cells, revealing a significant decrease in total m6A mRNA expression in cells after DSS treatment, while m6A levels were effectively increased in cells with METTL14 upregulation (Fig. 3A). Subsequently, the SRAMP program identified potential m6A modification sites on GPX4 mRNA (Fig. 3B). MeRIP-qPCR validated a marked elevation in m6A modification levels of GPX4 mRNA in METTL14-overexpressing NCM460 cells compared to the oe-NC group (Fig. 3C). To explore the m6A modification site on GPX4, we selected three very high confidence sites and establish the wild-type and mutant GPX4 reporter gene for each. As shown in Fig. 3D, overexpression of METTL14 significantly increased the luciferase activity in the WT group with site 1, while there were small differences in luciferase activity for sites 2 and 3. The results indicated that the binding between METTL14 and GPX4 was dependent on the m6A modification site at site 1. In addition, METTL14 overexpression significantly prolonged the half-life of GPX4 mRNA in contrast to control cells treated with actinomycin D (Fig. 3E). These findings indicated that METTL14 promoted GPX4 mRNA stability through mediated m6A modification.

Fig. 3
figure 3

METTL14 overexpression promoted the mRNA stability of GPX4. A The total level of m6A was quantified by m6A RNA methylation quantification kit. B SRAMP database (http://www.cuilab.cn/sramp) predicted the existence of multiple potential binding sites for GPX4 m6A modification. C The m6A modification status of GPX4 mRNA was determined using methylated RNA immunoprecipitation coupled with quantitative PCR (MeRIP-qPCR). D The m6A modification site of METTL14 on GPX4 mRNA was explored using a luciferase reporter. E The stability of GPX4 mRNA was examined by qRT-PCR assay. *p < 0.05, **p < 0.01, ***p < 0.001

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Silencing GPX4 partially reversed the inhibitory effect of METTL14 overexpression on ferroptosis in DSS-induced NCM460 cells

The aforementioned study has validated METTL14 role in regulating m6A methylation modification of GPX4. Following this, specific in vitro experiments will investigate their precise regulatory association. Using cell transfection methods, NCM460 cells treated with DSS were transfected with a specific short hairpin RNA (sh-GPX4) to suppress GPX4 expression. The efficacy of transfection was subsequently confirmed through qRT-PCR and Western blot (Fig. 4A, B). Knocking down GPX4 in DSS-treated NCM460 cells significantly impeded the enhanced cell viability resulting from high METTL14 expression (Fig. 4C). In addition, the levels of key markers closely related to ferroptosis such as ROS, GSH, MDA, and iron exhibited a substantial partial reversal in the upregulation or downregulation pattern induced by oe-METTL14 following the targeted GPX4 knockdown (Fig. 4D–G). This discovery implied that METTL14 might indirectly modulate cellular sensitivity to ferroptosis by controlling the m6A methylation modification of GPX4.

Fig. 4
figure 4

GPX4 inhibition partly reversed the effects of METTL14 on ferroptosis in DSS-treated NCM460 cells. A, B Transfection efficiency of GPX4 was assessed using qRT-PCR and western blot. C Cell viability in DSS-treated NCM460 cells was determined by CCK-8. D ROS levels in NCM460 cells treated with DSS were assessed using the ROS assay kit. E–G The levels of GSH (E), MDA (F), and iron (G) in NCM460 cells from various treatment groups were assessed using the GSH, MDA, and iron assay kits. *p < 0.05, **p < 0.01, ***p < 0.001

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METTL14 overexpression attenuated DSS-treated inflammatory bowel disease via upregulating GPX4 and inhibiting ferroptosis

We then delved into the precise mechanism by which METTL14 controlled GPX4 through in vivo experiments. Using the same methodology applied in the mouse trials detailed previously, we induced colitis in mice with DSS to create a colitis model. Subsequently, adeno-associated virus transduction was employed to enhance METTL14 expression in the intestinal mucosa of the mice. The DAI serves as a critical tool for evaluating disease activity, particularly in assessing the severity of intestinal conditions like colitis. Moreover, the reduction in colon length is a macroscopic indicator of UC. We observed a notable rise in DAI scores following DSS induction in the colitis model groups, contrasting with a score of 0 in the control group. Notably, all mice in the DSS group displayed considerably shorter colons than those in the control group, confirming the effectiveness of the model. Upregulation of METTL14 mitigated the escalation of DAI scores in colitis-afflicted mice, with the introduction of sh-GPX4 partially counteracting the aforementioned outcomes (Fig. 5A, D, and F). Subsequently, we assessed the expression levels of inflammatory factors and observed a significant increase in TNF-α and IL-1β expression in the inflamed mice. This increase was alleviated by the introduction of oe-METTL14, while sh-GPX4 significantly countered the downward trend induced by oe-METTL14 (Fig. 5B, C). Subsequently, we performed histopathological examination of colon tissues using HE staining. Mice in the control groups displayed preserved colon mucosal barriers and glands, typical crypt structures, and were devoid of ulcers, necrosis, or inflammatory cell infiltration. Conversely, mice in the DSS-treated groups exhibited marked colon mucosal injury, impaired glands, crypt loss, extensive infiltration of inflammatory cells, ulceration, and elevated histological inflammation scores (Fig. 5E). The oe-METTL14 group significantly ameliorated the aforementioned pathological findings. However, sh-GPX4 reversed the beneficial effects of oe-METTL14 on mouse intestinal inflammation (Fig. 5E). In addition, we assessed the expression of iron death-related markers in the mouse intestines. It was evident that the reduced GSH, GPX4 proteins and mRNA levels resulting from DSS treatment, as well as the elevated MDA and iron concentrations, were alleviated by oe-METTL14. However, sh-GPX4 partly mitigated these changes in trends (Fig. 5G–K). These results align with the outcomes of the in vitro functional experiments depicted in Fig. 4. The above conclusions indicate the significant involvement of METTL14 in reducing intestinal inflammation, preventing iron-related cell death, and highlight the substantial collaborative function of GPX4 regulation in this course.

Fig. 5
figure 5

METTL14 overexpression attenuated DSS-treated IBD via upregulating GPX4 and inhibiting ferroptosis. Mice were treated with (1) control, (2) DSS, (3) DSS + oe-NC, (4) DSS + oe-METTL14, (5) DSS + oe-METTL14 + sh-NC, (6) DSS + oe-METTL14 + sh-GPX4. A DAI scores of different groups of mice. B, C Serum TNF-α (B) and IL-1β (C) were analyzed by ELISA. D Colonic images of mice from different treatment groups. E The histopathology and histologic inflammatory scores of colitis mice were detected by hematoxylin–eosin staining. F The colon length in each group was measured. G–I GSH (G), MDA (H), and iron (I) in the colon tissues were estimated using the GSH, MDA, and iron assay kits. J, K The mRNA and protein of GPX4 in the colon tissues were analyzed using qRT-PCR and western blot. *p < 0.05, **p < 0.01, ***p < 0.001

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Discussion

Previous studies have emphasized the involvement of m6A methylation in the post-transcriptional regulation of gene expression in the pathogenesis of UC [32]. METTL14 is a key m6A methyltransferase [33]. However, the involvement of METTL14-mediated ferroptosis in UC remains largely unknown. Hence, this study provided an initial investigation into the precise functions and mechanisms of m6A in the onset and progression of inflammation in UC. In this study, we successfully established UC mice model using DSS induction, characterized by a notable decrease in colon length and an elevation in DAI score. These features align with the results of previously established models in earlier studies [34]. HE staining further illustrated extensive colonic tissue damage triggered by DSS treatment. Moreover, DSS exposure substantially heightened the inflammatory cytokines TNF-α and IL-1β levels in the colonic tissues of mice. Our investigation unveiled a notable decrease in METTL14 mRNA and protein expression in DSS-induced UC mice and NCM460 cells. In addition, Ji et al. found a reduced level of METTL14 mRNA in their analysis of m6A methylation-related genes in rats with colitis [35]. This conclusion derived from this research, mirroring the aforementioned findings, hints at the potential involvement of METTL14 in UC pathogenesis.

We also found that METTL14 overexpression has a suppression of the reduction of cell viability in DSS-induced NCM460 cells and induce GPX4 protein expression. GPX4 functioned as an antioxidant enzyme and was recognized as a pivotal factor in governing ferroptosis [36]. Prior literature has validated that modulating ferroptosis-related gene GPX4 expression, in conjunction with NOX1, can alleviate chemotherapy responsiveness in colorectal cancer [37]. This underlined the intimate correlation between ferroptosis and UC pathogenesis. Therefore, we subsequently explored the impact of changes in METTL14 expression on the levels of markers associated with ferroptosis. These markers included ROS, GSH, MDA, and iron. The excessive release of ROS associated with ferroptosis could disturb the body’s oxidation–antioxidation equilibrium, culminating in oxidative injury to the intestinal cells [38]. Due to oxidation reactions, polyunsaturated fatty acids in cell membrane phospholipids can produce a substantial quantity of lipid peroxidation byproducts like MDA, compromising cell membrane integrity and instigating ferroptosis [39]. Antioxidant enzymes such as GSH are vital in preventing tissue damage caused by ROS. Our research has shown that the upregulation of METTL14 reduced the levels of ROS, increased GSH levels, and decreased the concentrations of MDA and iron ions in DSS-induced NCM460 cells by promoting GPX4 expression. Rescue experiments have demonstrated that knocking down GPX4 partially reversed the inhibitory effect of overexpressed METTL14 on ferroptosis, including ROS, MDA, and iron levels, in DSS-induced NCM460 cells, while also partially reversing the promoting effect on GSH. Consequently, targeting ferroptosis could emerge as a new therapeutic avenue for treating UC.

We validated through the online prediction website SRAMP that the METTL14 mRNA sequence contained m6A modification sites. Through MeRIP-qPCR and RNA stability experiments, we confirmed that METTL14 orchestrated GPX4 mRNA expression in an m6A-dependent manner. The investigation into m6A modification extended into the realm of intestinal inflammatory conditions. For instance, METTL3/ALKBH5/YTHDF2 regulated the expression of KLF4 by mediating m6A modifications, thereby exacerbating the progression of inflammatory bowel disease [40]. YTHDF1 targeted Traf6 in an m6A-dependent fashion, modulating its translation processes and bolstering immune defenses during bacterial infections in the intestines [41]. Overall, these inquiries have broadened the horizons of m6A modifications, offering novel insights for exploring intestinal inflammation. Furthermore, through comprehensive animal studies, we further validated the pivotal role of METTL14 in alleviating intestinal inflammation and inhibiting ferroptosis, while also revealing its significant effect on the regulation of GPX4 expression.

This study has several limitations, primarily related to the sample size and the diversity of the participant group. These factors may affect the comprehensiveness and generalizability of the findings. Future research should seek to expand the sample size and conduct a more thorough exploration of additional regulatory pathways to formulate more precise treatment strategies for personalized therapy.

Conclusions

METTL14 upregulation relieved the clinical symptoms and inflammatory response in DSS-induced UC mice, enhancing the antioxidant enzyme GPX4 expression. Mechanistic investigation revealed that METTL14 regulated GPX4 mRNA expression and ferroptosis in an m6A-dependent manner (Fig. 6). This study will provide new theoretical foundations for understanding the pathogenesis and targeted therapies of UC.

Fig. 6
figure 6

Possible potential molecular mechanism diagram of METTL14 in UC. In cell and mouse models of DSS-induced colitis, GPX4 mRNA regulated ferroptosis in an m6A-dependent manner and influenced the development of UC. Mechanistic investigation unveiled that METTL14 identified the m6A modification of GPX4, regulating the stability of its mRNA and thus inhibiting ferroptosis

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Availability of data and materials

The data are available upon reasonable request from the corresponding author Ying Zhou.

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Author notes

  1. Yuhua Chen and Weicong Fan have contributed equally to this work.

  2. Jingsheng Liao and Ying Zhou are should be considered as the joint corresponding authors.

Authors and Affiliations

  1. Anorectal Department, Dongguan Hospital of Traditional Chinese Medicine, 1st Floor, No. 3, Dongcheng Section, Songshanhu Avenue, Dongcheng Street, Dongguan, 523000, Guangdong, China

    Yuhua Chen, Weicong Fan, Ying Lyu & Ying Zhou

  2. Medical Oncology, The Tenth Affiliated Hospital of Southern Medical University (Dongguan People’s Hospital), No. 78 Wandao Road, Dongguan, 523059, Guangdong, China

    Jingsheng Liao

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Contributions

Yuhua Chen and Weicong Fan designed the study and writing original draft. Ying Lyu collected data, processed statistical data, and performed the experiments. Ying Zhou and Jingsheng Liao designed, supervised and revised the study. All the authors reviewed the results and approved the final version of the manuscript.

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Chen, Y., Fan, W., Lyu, Y. et al. METTL14 modulates the progression and ferroptosis of colitis by regulating the stability of m6A-modified GPX4. Eur J Med Res 30, 88 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40001-025-02334-8

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European Journal of Medical Research

ISSN: 2047-783X

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