METTL3-mediated m6A Modification Research Articles

METTL3 restricts RIPK1-dependent cell death via the ATF3-cFLIP axis in the intestinal epithelium

Intestinal epithelial cells (IECs) are pivotal for maintaining intestinal homeostasis through self-renewal, proliferation, differentiation, and regulated cell death. While apoptosis and necroptosis are recognized as distinct pathways, their intricate interplay remains elusive. In this study, we report that Mettl3-mediated m6A modification maintains intestinal homeostasis by impeding epithelial cell death. Mettl3 knockout induces both apoptosis and necroptosis in IECs. Targeting different modes of cell death with specific inhibitors unveils that RIPK1 kinase activity is critical for the cell death triggered by Mettl3 knockout. Mechanistically, this occurs via the m6A-mediated transcriptional regulation of Atf3, a transcription factor that directly binds to Cflar, the gene encoding the anti-cell death protein cFLIP. cFLIP inhibits RIPK1 activity, thereby suppressing downstream apoptotic and necroptotic signaling. Together, these findings delineate the essential role of the METTL3-ATF3-cFLIP axis in homeostatic regulation of the intestinal epithelium by blocking RIPK1 activity.

N6-methyladenosine-modified SRPK1 promotes aerobic glycolysis of lung adenocarcinoma via PKM splicing

BackgroundThe RNA N6-methyladenosine (m6A) modification has become an essential hotspot in epigenetic modulation. Serine–arginine protein kinase 1 (SRPK1) is associated with the pathogenesis of various cancers. However, the m6A modification of SRPK1 and its association with the mechanism of in lung adenocarcinoma (LUAD) remains unclear.MethodsWestern blotting and polymerase chain reaction (PCR) analyses were carried out to identify gene and protein expression. m6A epitranscriptomic microarray was utilized to the assess m6A profile. Loss and gain-of-function assays were carried out elucidate the impact of METTL3 and SRPK1 on LUAD glycolysis and tumorigenesis. RNA immunoprecipitation (RIP), m6A RNA immunoprecipitation (MeRIP), and RNA stability tests were employed to elucidate the SRPK1’s METTL3-mediated m6A modification mechanism in LUAD. Metabolic quantification and co-immunoprecipitation assays were applied to investigate the molecular mechanism by which SRPK1 mediates LUAD metabolism.ResultsThe epitranscriptomic microarray assay revealed that SRPK1 could be hypermethylated and upregulated in LUAD. The main transmethylase METTL3 was upregulated and induced the aberrant high m6A levels of SRPK1. Mechanistically, SRPK1’s m6A sites were directly methylated by METTL3, which also stabilized SRPK1 in an IGF2BP2-dependent manner. Methylated SRPK1 subsequently promoted LUAD progression through enhancing glycolysis. Further metabolic quantification, co-immunoprecipitation and western blot assays revealed that SRPK1 interacts with hnRNPA1, an important modulator of PKM splicing, and thus facilitates glycolysis by upregulating PKM2 in LUAD. Nevertheless, METTL3 inhibitor STM2457 can reverse the above effects in vitro and in vivo by suppressing SRPK1 and glycolysis in LUAD.ConclusionIt was revealed that in LUAD, aberrantly expressed METTL3 upregulated SRPK1 levels via an m6A-IGF2BP2-dependent mechanism. METTL3-induced SRPK1 fostered LUAD cell proliferation by enhancing glycolysis, and the small-molecule inhibitor STM2457 of METTL3 could be an alternative novel therapeutic strategy for individuals with LUAD.

METTL3 aggravates cell damage induced by Streptococcus pneumoniae via the NEAT1/CTCF/MUC19 axis.

Disruption of the alveolar barrier can trigger acute lung injury. This study elucidated the association of methyltransferase-like 3 (METTL3) with Streptococcus pneumoniae (SP)-induced apoptosis and inflammatory injury of alveolar epithelial cells (AECs). AECs were cultured and then infected with SP. Furthermore, the expression of METTL3, interleukin (IL)-10, IL-6, tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1), mucin 19 (MUC19), N6-methyladenosine (m6A), and NEAT1 after m6A modification were detected by qRT-PCR, Western blot, and enzyme-linked immunosorbent, m6A quantification, and methylated RNA immunoprecipitation-qPCR analyses, respectively. Moreover, the subcellular localization of NEAT1 was analyzed by nuclear/cytosol fractionation assay, and the binding between NEAT1 and CCCTC-binding factor (CTCF) was also analyzed. The results of this investigation revealed that SP-induced apoptosis and inflammatory injury in AECs and upregulated METTL3 expression. In addition, the downregulation of METTL3 alleviated apoptosis and inflammatory injury in AECs. METTL3-mediated m6A modification increased NEAT1 and promoted its binding with CTCF to facilitate MUC19 transcription. NEAT1 or MUC19 overexpression disrupted their protective role of silencing METTL3 in AECs, thereby increasing apoptosis and inflammatory injury. In conclusion, this is the first study to suggest that METTL3 aggravates SP-induced cell damage via the NEAT1/CTCF/MUC19 axis.

MiR-340-3p-modified bone marrow mesenchymal stem cell-derived exosomes inhibit ferroptosis through METTL3-mediated m6A modification of HMOX1 to promote recovery of injured rat uterus

BackgroundFerroptosis is associated with the pathological progression of hemorrhagic injury and ischemia–reperfusion injury. According to our previous study, exosomes formed through bone marrow mesenchymal stem cells modified with miR-340-3p (MB-exos) can restore damaged endometrium. However, the involvement of ferroptosis in endometrial injury and the effect of MB-exos on ferroptosis remain elusive.MethodsThe endometrial injury rat model was developed. Exosomes were obtained from the supernatants of bone marrow mesenchymal stromal cells (BMSCs) and miR-340/BMSCs through differential centrifugation. We conducted RNA-seq analysis on endometrial tissues obtained from the PBS and MB-exos groups. Ferroptosis was induced in endometrial stromal cells (ESCs) by treating them with erastin or RSL3, followed by treatment with B-exos or MB-exos. We assessed the endometrial total m6A modification level after injury and subsequent treatment with B-exos or MB-exos by methylation quantification assay. We performed meRIP-qPCR to analyze m6A modification-regulated endogenous mRNAs.ResultsWe reveal that MB-exos facilitate the injured endometrium to recover by suppressing ferroptosis in endometrial stromal cells. The injured endometrium showed significantly upregulated N6-methyladenosine (m6A) modification levels; these levels were attenuated by MB-exos through downregulation of the methylase METTL3. Intriguingly, METTL3 downregulation appears to repress ferroptosis by stabilizing HMOX1 mRNA, thereby potentially elucidating the mechanism through which MB-exos inhibit ferroptosis in ESCs. We identified YTHDF2 as a critical m6A reader protein that contributes to HMOX1 mRNA degradation. YTHDF2 facilitates HMOX1 mRNA degradation by identifying the m6A binding site in the 3′-untranslated regions of HMOX1. In a rat model, treatment with MB-exos ameliorated endometrial injury-induced fibrosis by inhibiting ferroptosis in ESCs. Moreover, METTL3 short hairpin RNA-mediated inhibition of m6A modification enhanced the inhibitory effect of MB-exos on ferroptosis in endometrial injury.ConclusionsThus, these observations provide new insights regarding the molecular mechanisms responsible for endometrial recovery promotion by MB-exos and highlight m6A modification-dependent ferroptosis inhibition as a prospective therapeutic target to attenuate endometrial injury.

Qianggu Decoction Alleviated Osteoporosis by Promoting Osteogenesis of BMSCs through Mettl3-Mediated m6A Methylation.

Osteoporosis development is linked to abnormal bone marrow mesenchymal stem cells (BMSCs) differentiation. N6-methyladenosine (m6A), a prevalent mRNA modification, is known to influence BMSCs' osteogenic capacity. Qianggu decoction (QGD), a traditional Chinese medicine for osteoporosis, has unknown effects on BMSCs differentiation. This study investigates QGD's impact on BMSCs and its potential to ameliorate osteoporosis through m6A regulation. Using Sprague-Dawley (SD) rats with ovariectomy-induced osteoporosis, it is evaluated QGD's antiosteoporotic effects through micro-CT, histology, Western blotting, and osteoblastogenesis markers. QGD is found to enhance bone tissue growth and upregulate osteogenic markers Runx2, OPN, and OCN. It also promoted BMSCs osteogenic differentiation, as shown by increased calcium nodules and ALP activity. QGD treatment significantly increased m6A RNA levels and Mettl3 expression in BMSCs. Silencing Mettl3 with siRNA negated QGD's osteogenic effects. Collectively, QGD may improve BMSCs differentiation and mitigate osteoporosis, potentially through Mettl3-mediated m6A modification.

METTL3-m6A methylation inhibits the proliferation and viability of type II alveolar epithelial cells in acute lung injury by enhancing the stability and translation efficiency of Pten mRNA

BackgroundThe pathogenesis of acute lung injury (ALI) involves a severe inflammatory response, leading to significant morbidity and mortality. N6-methylation of adenosine (m6A), an abundant mRNA nucleotide modification, plays a crucial role in regulating mRNA metabolism and function. However, the precise impact of m6A modifications on the progression of ALI remains elusive.MethodsALI models were induced by either intraperitoneal injection of lipopolysaccharide (LPS) into C57BL/6 mice or the LPS-treated alveolar type II epithelial cells (AECII) in vitro. The viability and proliferation of AECII were assessed using CCK-8 and EdU assays. The whole-body plethysmography was used to record the general respiratory functions. M6A RNA methylation level of AECII after LPS insults was detected, and then the “writer” of m6A modifications was screened. Afterwards, we successfully identified the targets that underwent m6A methylation mediated by METTL3, a methyltransferase-like enzyme. Last, we evaluated the regulatory role of METTL3-medited m6A methylation at phosphatase and tensin homolog (Pten) in ALI, by assessing the proliferation, viability and inflammation of AECII.ResultsLPS induced marked damages in respiratory functions and cellular injuries of AECII. The m6A modification level in mRNA and the expression of METTL3, an m6A methyltransferase, exhibited a notable rise in both lung tissues of ALI mice and cultured AECII cells subjected to LPS treatment. METTL3 knockdown or inhibition improved the viability and proliferation of LPS-treated AECII, and also reduced the m6A modification level. In addition, the stability and translation of Pten mRNA were enhanced by METTL3-mediated m6A modification, and over-expression of PTEN reversed the protective effect of METTL3 knockdown in the LPS-treated AECII.ConclusionsThe progression of ALI can be attributed to the elevated levels of METTL3 in AECII, as it promotes the stability and translation of Pten mRNA through m6A modification. This suggests that targeting METTL3 could offer a novel approach for treating ALI.

METTL3-mediated m6A Modification of hsa_circ_0131922 Attenuates the Progression of Papillary Thyroid Cancer by Regulating the p53 Pathway.

This study aimed to confirm the regulatory role and mechanism of circular RNA (circRNA) hsa_circ_0131922 in Papillary Thyroid Carcinoma (PTC) progression. Accumulating evidence suggests that N6-methyladenosine (m6A)-modified circular RNAs (circRNAs) perform pivotal functions in various malignancies. However, the specific role of the m6A modification of circRNA mediated by METTL3 in Papillary Thyroid Carcinoma (PTC) remains undocumented. In this work, we aimed to examine the molecular mechanisms of a novel m6Amodified circRNA, hsa_circ_0131922, in PTC progression. Potential circRNA was identified from GEO datasets. The RNA or protein levels of hsa_circ_0131922, METTL3, p53, and p21 were evaluated by qRT-PCR or western blot assays. The various cellular functions were checked by CCK8, wound healing, transwell, and xenograft tumor assays. MeRIP-qPCR was performed to observe the METTL3-mediated m6A modification of hsa_circ_0131922. Furthermore, the interactions between hsa_circ_0131922 and METTL3 in PTC were analyzed by bioinformatics analysis and various rescue experiments. The levels of hsa_circ_0131922 were markedly downregulated in PTC tissues and cell lines. In addition, the lower hsa_circ_0131922 levels correlated with poor prognosis in PTC patients. The hsa_circ_0131922 overexpression reduced the malignant phenotypes of PTC cells and activated the p53/p21 pathway. Bioinformatic analysis showed the m6A-modified sites of hsa_circ_0131922, and a positive correlation between hsa_circ_0131922 and METTL3. Moreover, overexpression of METTL3 increased the levels of m6A modification of hsa_circ_0131922. Mechanistically, the anti-tumor effects of hsa_circ_0131922 overexpression have been found to be partially reversed by silencing METTL3 in vivo and in vitro. The results have demonstrated m6A-modified hsa_circ_0131922 by METTL3 to attenuate the progression of PTC by regulating the p53 pathway. Therefore, hsa_circ_0131922 could be a predictive prognostic biomarker and therapeutic target for PTC.

METTL3-mediated NDUFB5 m6A modification promotes cell migration and mitochondrial respiration to promote the wound healing of diabetic foot ulcer

BackgroundDiabetic foot ulcer (DFU) is the most devastating complication of diabetes mellitus (DM) and plays a major role in disability and death in DM patients. NADH: ubiquinone oxidoreductase subunit B5 (NDUFB5) plays an important role in maintaining mitochondrial respiration, but whether it is involved in regulating the progression of advanced glycation end products (AGEs)-mediated DFU is still unclear.MethodsFirstly, the role of AGEs on cell viability, migration, and mitochondrial respiration in human umbilical vein endothelial cells (HUVECs) was explored in vitro. Next, NDUFB5 expression was detected in human samples and AGEs-treated HUVECs, and NDUFB5’s effect on AGEs-induced HUVECs injury and skin wound in diabetic mice was further clarified. In addition, the role of m6A modification mediated by methyltransferase-like 3 (METTL3) in regulating NDUFB5 expression and AGEs-induced HUVECs injury was investigated.ResultsNDUFB5 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs, whereas mitochondrial fusion promoter M1 facilitated cell viability, migration, and mitochondrial oxiadative respiration in NDUFB5 knockdown HUVECs. Meanwhile, NDUFB5 promotes skin wound healing in diabetic mice. Besides, METTL3-mediated m6A modification and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) enhanced NDUFB5 expression in HUVECs. Furthermore, METTL3 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs by increasing NDUFB5.ConclusionMETTL3-mediated NDUFB5 m6A modification inhibits AGEs-induced cell injury in HUVECs. METTL3 and NDUFB5 might serve as potential targets for DFU therapy in the future.

M6A-mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8+ T cells and phagocytosis of macrophages.

Tumor immune evasion relies on the crosstalk between tumor cells and adaptive/innate immune cells. Immune checkpoints play critical roles in the crosstalk, and immune checkpoint inhibitors have achieved promising clinical effects. The long non-coding RNA taurine-upregulated gene 1 (TUG1) is upregulated in hepatocellular carcinoma (HCC). However, how TUG1 is upregulated and the effects on tumor immune evasion are incompletely understood. Here, METTL3-mediated m6A modification led to TUG1 upregulation is demonstrated. Knockdown of TUG1 inhibited tumor growth and metastasis, increased the infiltration of CD8+ T cells and M1-like macrophages in tumors, promoted the activation of CD8+ T cells through PD-L1, and improved the phagocytosis of macrophages through CD47. Mechanistically, TUG1 regulated PD-L1 and CD47 expressions by acting as a sponge of miR-141 and miR-340, respectively. Meanwhile, TUG1 interacted with YBX1 to facilitate the upregulation of PD-L1 and CD47 transcriptionally, which ultimately regulated tumor immune evasion. Clinically, TUG1 positively correlated with PD-L1 and CD47 in HCC tissues. Moreover, the combination of Tug1-siRNA therapy with a Pdl1 antibody effectively suppressed tumor growth. Therefore, the mechanism of TUG1 in regulating tumor immune evasion is revealed and can inform existing strategies targeting TUG1 for enhancing HCC immune therapy and drug development.

Involvement of METTL3-mediated m6A methylation in the early development of porcine cloned embryos

METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2, GATA3, NANOG and YAP, and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos.

METTL3-mediated N6-methyladenosine modification of STAT5A promotes gastric cancer progression by regulating KLF4

N6-methyladenosine (m6A) is the predominant post-transcriptional RNA modification in eukaryotes and plays a pivotal regulatory role in various aspects of RNA fate determination, such as mRNA stability, alternative splicing, and translation. Dysregulation of the critical m6A methyltransferase METTL3 is implicated in tumorigenesis and development. Here, this work showed that METTL3 is upregulated in gastric cancer tissues and is associated with poor prognosis. METTL3 methylates the A2318 site within the coding sequence (CDS) region of STAT5A. IGF2BP2 recognizes and binds METTL3-mediated m6A modification of STAT5A through its GXXG motif in the KH3 and KH4 domains, leading to increased stability of STAT5A mRNA. In addition, both METTL3 and IGF2BP2 are positively correlated with STAT5A in human gastric cancer tissue samples. Helicobacter pylori infection increased the expression level of METTL3 in gastric cancer cells, thereby leading to the upregulation of STAT5A. Functional studies indicated that STAT5A overexpression markedly enhances the proliferation and migration of GC cells, whereas STAT5A knockdown has inhibitory effects. Further nude mouse experiments showed that STAT5A knockdown effectively inhibits the growth and metastasis of gastric cancer in vivo. Moreover, as a transcription factor, STAT5A represses KLF4 transcription by binding to its promoter region. The overexpression of KLF4 can counteract the oncogenic impact of STAT5A. Overall, this study highlights the crucial role of m6A in gastric cancer and provides potential therapeutic targets for gastric cancer.

METTL3-Mediated N6-Methyladenosine mRNA Modification and cGAS-STING Pathway Activity in Kidney Fibrosis

Key Points METTL3 was upregulated in CKD, leading to increased m6A modification levels.M6A modifications were enriched in genes related to the cyclic guanosine monophosphate–AMP synthase-stimulator of IFN genes pathway in CKD.Normalizing heightened METTL3 and m6A modification levels showed promise as a strategy to combat kidney disease. Background Chemical modifications on RNA profoundly affect RNA function and regulation. m6A, the most abundant RNA modification in eukaryotes, plays a pivotal role in diverse cellular processes and disease mechanisms. However, its importance is understudied in human CKD samples regarding its influence on pathological mechanisms. Methods Liquid chromatography–tandem mass spectrometry and methylated RNA immunoprecipitation sequencing were used to examine alterations in m6A levels and patterns in CKD samples. Overexpression of the m6A writer METTL3 in cultured kidney tubular cells was performed to confirm the effect of m6A in tubular cells and explore the biological functions of m6A modification on target genes. In addition, tubule-specific deletion of Mettl3 (Ksp-Cre Mettl3f/f) mice and antisense oligonucleotides inhibiting Mettl3 expression were used to reduce m6A modification in an animal kidney disease model. Results By examining 127 human CKD samples, we observed a significant increase in m6A modification and METTL3 expression in diseased kidneys. Epitranscriptomic analysis unveiled an enrichment of m6A modifications in transcripts associated with the activation of inflammatory signaling pathways, particularly the cyclic guanosine monophosphate–AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway. m6A hypermethylation increased mRNA stability in cGAS and STING1 as well as elevated the expression of key proteins within the cGAS-STING pathway. Both the tubule-specific deletion of Mettl3 and the use of antisense oligonucleotides to inhibit Mettl3 expression protected mice from inflammation, reduced cytokine expression, decreased immune cell recruitment, and attenuated kidney fibrosis. Conclusions Our research revealed heightened METTL3-mediated m6A modification in fibrotic kidneys, particularly enriching the cGAS-STING pathway. This hypermethylation increased mRNA stability for cGAS and STING1, leading to sterile inflammation and fibrosis.

METTL3 promotes the osteogenic differentiation of human periodontal ligament cells by increasing YAP activity via IGF2BP1 and YTHDF1-mediated m6A modification.

N6-Methyladenosine (m6A) has been confirmed to play a dynamic role in osteoporosis and bone metabolism. However, whether m6A is involved in the osteogenic differentiation of human periodontal ligament cells (hPDLCs) remains unclear. The present study aimed to verify the role of methyltransferase-like 3 (METTL3)-mediated m6A modification in the osteogenic differentiation of hPDLCs. The METTL3, Runx2, Osx, and YAP mRNA expression was determined by qPCR. METTL3, RUNX2, OSX, YTHDF1, YAP, IGF2BP1, and eIF3a protein expression was measured by Western blotting and immunofluorescence assays. The levels of m6A modification were evaluated by methylated RNA immunoprecipitation (MeRIP) and dot blot analyses. MeRIP-seq and RNA-seq were used to screen potential candidate genes. Nucleic acid and protein interactions were detected by immunoprecipitation. Alizarin red staining was used to evaluate the osteogenic differentiation of hPDLCs. Gene transcription and promoter activities were assessed by luciferase reporter assays (n ≥ 3). The expression of METTL3 and m6A modifications increased synchronously with the osteogenic differentiation of hPDLCs (p = .0016). YAP was a candidate gene identified by MeRIP-seq and RNA-seq, and its mRNA and protein expression levels were simultaneously increased. METTL3 increased the m6A methylated IGF2BP1-mediated stability of YAP mRNA (p = .0037), which in turn promoted osteogenic differentiation (p = .0147). Furthermore, METTL3 increased the translation efficiency of YAP by recruiting YTHDF1 and eIF3a to the translation initiation complex (p = .0154), thereby promoting the osteogenic differentiation of hPDLCs (p = .0012). Our study revealed that METTL3-initiated m6A mRNA methylation promotes osteogenic differentiation of hPDLCs by increasing IGF2BP1-mediated YAP mRNA stability and recruiting YTHDF1 and eIF3a to the translation initiation complex to increase YAP mRNA translation. Our findings reveal the mechanism of METTL3-mediated m6A modification during hPDLC osteogenesis, providing a potential therapeutic target for periodontitis and alveolar bone defects.

METTL3's role in cervical cancer development through m6A modification.

N6-methylated adenosine (m6A) is a crucial RNA modification in eukaryotes, particularly in cancer. However, its role in cervical cancer (CC) is unclear. We aimed to elucidate the part of m6A in CC by analyzing methyltransferase-like 3 (METTL3) expression, identifying downstream targets, and exploring the underlying mechanism. We assessed METTL3 expression in CC using western blotting, quantitative polymerase chain reaction (qPCR), and immunohistochemistry. Invitro and invivo experiments examined METTL3's role in CC. We employed RNA sequencing, methylated RNA immunoprecipitation sequencing, qPCR, and RNA immunoprecipitation qPCR to explore METTL3's mechanism in CC. METTL3 expression was upregulated in CC, promoting cell proliferation and metastasis. METTL3 knockdown inhibited human cervical cancer by inactivating AKT/mTOR signaling pathway. METTL3-mediated m6A modification was observed in CC cells, targeting phosphodiesterase 3A (PDE3A). METTL3 catalyzed m6A modification on PDE3A mRNA through YTH domain family protein 3 (YTHDF3). Our study indicated the mechanism of m6A modification in CC and suggested the METTL3/YTHDF3/PDE3A axis as a potential clinical target for CC treatment.

TRPM4 mRNA stabilization by METTL3-mediated m6A modification promotes calcific aortic valve inflammation

BackgroundTransient receptor potential melastatin 4 (TRPM4) affects immune responses by regulating calcium homeostasis, but its role in calcific aortic valve inflammation remains unclear. This study aimed to assess the expression and function of TRPM4 in patients with or without calcific aortic valve disease (CAVD). MethodsThe mRNA and protein expression levels of TRPM4 and related factors in calcified and noncalcified tissues were measured using qRT-PCR and Western blot. The proteins interacting with TRPM4 were confirmed by RNA pull-down and RNA immunoprecipitation assays. Dual-Luciferase Reporter Assay was performed to confirm the m6A site of TRPM4. ResultsThe mRNA expression levels of TRPM4, TLR4, IL-6, MCP-1, TNF-α, and NF-κB p65 were significantly higher in calcified aortic valve tissues than in noncalcified tissues, and TRPM4 was significantly positively correlated with inflammation-related factors. The protein expression level of TRPM4, TLR4 and NF-κB p65 were significantly higher in calcified aortic valve tissues than in noncalcified tissues. N6-methyladenosine (m6A) modification of TRPM4 mRNA by METTL3-YTHDF1 up-regulated its expression in CAVD. And TRPM4 promoted the level of inflammation via activation of the JNK-MAPK signaling pathway, after knockdown TRPM4, the production of proinflammatory cytokines was significantly suppressed. ConclusionThe results indicate the pivotal role of TRPM4 in CAVD and highlight METTL3-mediated m6A modification of TRPM4 in promoting inflammation through JNK-MAPK signaling pathway. This work provides potential therapeutic strategy to impede inflammation in CAVD.

METTL3-dependent m6A modification facilitates decreased endometrial decidualization via attenuation of MET in endometriosis.

Failure to induce mesenchymal-epithelial transition (MET) during stromal cell decidualization can lead to consequences such as impaired fertility in patients with endometriosis. METTL3-mediated m6A modification plays an important role in attenuating MET and defective decidualization of endometrial stromal cells and contributes to the development of reduced endometrial receptivity in endometriosis. Mesenchymal-epithelial transition (MET)-mediated endometrial decidualization is pivotal for achieving endometrial receptivity and successful pregnancy. We observed blockade of MET in the eutopic secretory endometrium of patients with endometriosis, but the underlying mechanism is unknown. In this study, real-time PCR was used to detect PRL and IGFBP1 expression, whereas western blotting was used to detect the expression of MET markers and METTL3. Phalloidin staining was used to identify changes in cell morphology. M6A levels were quantified using a colorimetric method and m6A dot blots, and functional analysis was performed using spheroid adhesion assays. We first found that increased E-cadherin expression was accompanied by decreased vimentin and Slug expression in the eutopic secretory endometrium of individuals with endometriosis. We also detected a significant increase in both the m6A level and the expression of the related methyltransferase METTL3. Finally, METTL3 expression was negatively correlated with PRL, IGFBP1, and MET markers expression. Collectively, our findings suggest that METTL3 mediates m6A modification, thereby inhibiting MET formation within the eutopic secretory endometrium of patients with endometriosis. Increased METTL3-mediated m6A modification plays a crucial role in attenuating MET formation and decidualization impairment in endometrial stromal cells, ultimately contributing to compromised endometrial receptivity in individuals with endometriosis. These insights could lead to the identification of potential therapeutic targets for improving both endometrial receptivity and pregnancy rate among individuals affected by endometriosis.

Histone lactylation-regulated METTL3 promotes ferroptosis via m6A-modification on ACSL4 in sepsis-associated lung injury

Elevated lactate levels are a significant biomarker of sepsis and are positively associated with sepsis-related mortality. Sepsis-associated lung injury (ALI) is a leading cause of poor prognosis in clinical patients. However, the underlying mechanisms of lactate's involvement in sepsis-associated ALI remain unclear. In this study, we demonstrate that lactate regulates N6-methyladenosine (m6A) modification levels by facilitating p300-mediated H3K18la binding to the METTL3 promoter site. The METTL3-mediated m6A modification is enriched in ACSL4, and its mRNA stability is regulated through a YTHDC1-dependent pathway. Furthermore, short-term lactate stimulation upregulates ACSL4, which promotes mitochondria-associated ferroptosis. Inhibition of METTL3 through knockdown or targeted inhibition effectively suppresses septic hyper-lactate-induced ferroptosis in alveolar epithelial cells and mitigates lung injury in septic mice. Our findings suggest that lactate induces ferroptosis via the GPR81/H3K18la/METTL3/ACSL4 axis in alveolar epithelial cells during sepsis-associated ALI. These results reveal a histone lactylation-driven mechanism inducing ferroptosis through METTL3-mediated m6A modification. Targeting METTL3 represents a promising therapeutic strategy for patients with sepsis-associated ALI.

M6A-induced transcription factor IRF5 contributes to the progression of cervical cancer by upregulating PPP6C.

Cervical cancer (CC) is a gynaecological malignancy tumour that seriously threatens women's health. Recent evidence has identified that interferon regulatory factor 5 (IRF5), a nucleoplasm shuttling protein, is a pivotal transcription factor regulating the growth and metastasis of various human tumours. This study aimed to investigate the function and molecular basis of IRF5 in CC development. IRF5, protein phosphatase 6 catalytic subunit (PPP6C) and methyltransferase-like 3 (METTL3) mRNA levels were evaluated by quantitative real-time (qRT)-polymerase chain reaction (PCR). IRF5, PPP6C, METTL3, B-cell lymphoma 2 and Bax protein levels were detected using western blot. Cell proliferation, migration, invasion, angiogenesis and apoptosis were determined by using colony formation, 5-ethynyl-2'-deoxyuridine (EdU), transwell, tube formation assay and flow cytometry assay, respectively. Glucose uptake and lactate production were measured using commercial kits. Xenograft tumour assay in vivo was used to explore the role of IRF5. After JASPAR predication, binding between IRF5 and PPP6C promoter was verified using chromatin immunoprecipitation and dual-luciferase reporter assays. Moreover, the interaction between METTL3 and IRF5 was verified using methylated RNA immunoprecipitation (MeRIP). IRF5, PPP6C and METTL3 were highly expressed in CC tissues and cells. IRF5 silencing significantly inhibited cell proliferation, migration, invasion, angiogenesis and glycolytic metabolism in CC cells, while induced cell apoptosis. Furthermore, the absence of IRF5 hindered tumour growth in vivo. At the molecular level, IRF5 might bind with PPP6C to positively regulate the expression of PPP6C mRNA. Meanwhile, IRF5 was identified as a downstream target of METTL3-mediated m6A modification. METTL3-mediated m6A modification of mRNA might promote CC malignant progression by regulating PPP6C, which might provide a promising therapeutic target for CC treatment.

Berberine inhibits the progression of breast cancer by regulating METTL3-mediated m6A modification of FGF7 mRNA.

Berberine (BBR), an isoquinoline alkaloid from Coptidis rhizoma, has been found to have powerful activities against various human malignancies, including breast cancer. However, the underlying antitumor mechanisms of BBR in breast cancer remain poorly understood. Breast cancer cells were cultured and treated with different doses (0, 20, 40, and 60 μM) of BBR for 48 h. Cell viability, proliferation, apoptosis, invasion, and migration were assessed using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and wound healing assays. Fibroblast growth factor 7 (FGF7), methyltransferase-like 3 (METTL3), and insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) mRNA levels and protein levels were measured using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Interaction between METTL3 and FGF7 m6A was assessed using methylated RNA immunoprecipitation (MeRIP)-qPCR and RNA immunoprecipitation (RIP) assay. Binding ability between IGF2BP3 and FGF7 mRNA was analyzed using RIP assay. BBR treatment hindered breast cancer cell proliferation, invasion, migration, and induced apoptosis. FGF7 expression was upregulated in breast cancer tissues, while its level was reduced in BBR-treated tumor cells. FGF7 upregulation relieved the repression of BBR on breast cancer cell malignant behaviors. In mechanism, METTL3 stabilized FGF7 mRNA through the m6A-IGF2BP3-dependent mechanism and naturally improved FGF7 expression. BBR treatment inhibited breast cancer growth in vivo. BBR treatment blocked breast cancer cell growth and metastasis partly by regulating METTL3-mediated m6A modification of FGF7 mRNA, providing a promising therapeutic target for breast cancer treatment.

METTL3-mediated deficiency of lncRNA HAR1A drives non-small cell lung cancer growth and metastasis by promoting ANXA2 stabilization

We previously reported lncRNA HAR1A as a tumor suppressor in non-small cell lung cancer (NSCLC). However, the delicate working mechanisms of this lncRNA remain obscure. Herein, we demonstrated that the ectopic expression of HAR1A inhibited the proliferation, epithelial-mesenchymal transition (EMT), migration, and invasion of NSCLC cells and enhanced paclitaxel (PTX) sensitivity in vitro and in vivo. We identified the oncogenic protein annexin 2 (ANXA2) as a potential interacting patterner of HAR1A. HAR1A overexpression enhanced ANXA2 ubiquitination and accelerated its degradation via the ubiquitin–proteasome pathway. We further uncovered that HAR1A promoted the interaction between E3 ubiquitin ligase TRIM65 and ANXA2. Moreover, the ANXA2 plasmid transfection could reverse HAR1A overexpression-induced decreases in proliferation, migration, and invasion of NSCLC cells and the activity of the NF-κB signaling pathway. Finally, we found that HAR1A loss in NSCLC might be attributed to the upregulated METTL3. The m6A modification levels of HAR1A were increased in cancer cells, while YTHDF2 was responsible for recognizing m6A modification in the HAR1A, leading to the disintegration of this lncRNA. In conclusion, we found that METTL3-mediated m6A modification decreased HAR1A in NSCLC. HAR1A deficiency, in turn, stimulated tumor growth and metastasis by activating the ANXA2/p65 axis.