Methyltransferase-like 3 Research Articles

SALL4 promotes cancer stem-like cell phenotype and radioresistance in oral squamous cell carcinomas via methyltransferase-like 3-mediated m6A modification

Radioresistance imposes a great challenge in reducing tumor recurrence and improving the clinical prognosis of individuals having oral squamous cell carcinoma (OSCC). OSCC harbors a subpopulation of CD44(+) cells that exhibit cancer stem-like cell (CSC) characteristics are involved in malignant tumor phenotype and radioresistance. Nevertheless, the underlying molecular mechanisms in CD44( + )-OSCC remain unclear. The current investigation demonstrated that methyltransferase-like 3 (METTL3) is highly expressed in CD44(+) cells and promotes CSCs phenotype. Using RNA-sequencing analysis, we further showed that Spalt-like transcription factor 4 (SALL4) is involved in the maintenance of CSCs properties. Furthermore, the overexpression of SALL4 in CD44( + )-OSCC cells caused radioresistance in vitro and in vivo. In contrast, silencing SALL4 sensitized OSCC cells to radiation therapy (RT). Mechanistically, we illustrated that SALL4 is a direct downstream transcriptional regulation target of METTL3, the transcription activation of SALL4 promotes the nuclear transport of β-catenin and the expression of downstream target genes after radiation therapy, there by activates the Wnt/β-catenin pathway, effectively enhancing the CSCs phenotype and causing radioresistance. Herein, this study indicates that the METTL3/SALL4 axis promotes the CSCs phenotype and resistance to radiation in OSCC via the Wnt/β-catenin signaling pathway, and provides a potential therapeutic target to eliminate radioresistant OSCC.

The METTL3-m6A-YTHDC1-AMIGO2 axis contributes to cell proliferation and migration in esophageal squamous cell carcinoma

The upregulation of methyltransferase-like 3 (METTL3) has been associated with the progression of esophageal cancer. However, METTL3-induced N6-methyladenosine (m6A) alterations on the downstream target mRNAs in esophageal squamous cell carcinoma (ESCC) are not yet fully understood. Our study revealed that silencing METTL3 resulted in a significant decrease in ESCC cell proliferation and metastasis in vitro and in vivo. Additionally, the adhesion molecule with Ig like domain 2 (AMIGO2) was identified as a potential downstream target of both METTL3 and YTH Domain-Containing Protein 1 (YTHDC1) in ESCC cells. Functionally, AMIGO2 augmented the malignant behaviors of ESCC cells in vitro and in vivo, and its overexpression can rescue the inhibition of the proliferation and migration in ESCC cells induced by METTL3 or YTHDC1 knockdown. Furthermore, our findings revealed that knockdown of METTL3 decreased m6A modification in the 5′-untranslated regions (5′UTR) of AMIGO2 precursor mRNA (pre-mRNA), and YTHDC1 interacted with AMIGO2 pre-mRNA to regulate AMIGO2 expression by modulating the splicing process of AMIGO2 pre-mRNA in ESCC cells. These findings highlighted a novel role of the METTL3-m6A-YTHDC1-AMIGO2 axis in regulating ESCC cell proliferation and motility, suggesting its potential as a therapeutic target for ESCC.

METTL3-mediated methylation of CYP2C19 mRNA may aggravate clopidogrel resistance in ischemic stroke patients.

N6-methyladenosine (m6A) is the most frequently occurring interior modification in eukaryotic messenger RNA (mRNA), and abnormal mRNA modifications can affect many biological processes. However, m6A's effect on the metabolism of antiplatelet drugs for the prevention of ischemic stroke (IS) remains largely unclear. We analyzed the m6A enzymes and m6A methylation in peripheral blood samples of IS patients with/without clopidogrel resistance (CR), and the peripheral blood and liver of rat models with/without CR. We also compared the effect of m6A methylation on the expression of the drug-metabolizing enzymes (CYP2C19 and CYP2C6v1) in CR and non-CR samples. Methyltransferase-like 3 (METTL3), an m6A enzyme, was highly expressed in the peripheral blood of patients with CR, and in both the peripheral blood and liver of rats with CR. This enzyme targets CYP2C19 or CYP2C6v1 mRNA through m6A methylation, resulting in low expression of CYP2C19 or CYP2C6v1 mRNA. Consequently, this leads to decreased clopidogrel metabolism and CR. The METTL3-mediated methylation of CYP2C19 mRNA may aggravate CR in IS patients.

METTL1 facilitates ameloblastoma invasive growth via MAPK signaling pathway

ObjectivesAmeloblastoma (AM), a common odontogenic epithelial tumor, exhibits aggressive growth due to incomplete encapsulation within the jawbone. Postoperative recurrence is a significant concern, closely associated with its invasive nature. We investigate the role of tRNA N-7 methylguanosine (m7G) modification mediated by Methyltransferase-like 1 (METTL1) in AM's invasive growth and prognosis. Materials and MethodsMETTL1 expression was analyzed in diverse cell lines and clinical AM tissues. Its association with postoperative AM recurrence was examined. Functional experiments included METTL1 gene silencing using shRNA in hTERT-AM cells, assessing cell proliferation, migration, and invasion. Xenograft tumor model was constructed to investigate tumor growth. Molecular mechanisms behind METTL1′s role in AM invasiveness were elucidated using Ribosome nascent-chain complex-bound mRNA sequencing (RNC-seq) and experimental analysis. ResultsHigh METTL1 expression was significantly associated with postoperative recurrence in AM. The inhibition of AM development following METTL1 knockdown has been corroborated by experiments conducted both in vitro and in vivo. Analysis of RNC-seq data revealed that downregulated genes were predominantly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, suggesting that METTL1 may promote AM's invasive growth through the MAPK signaling pathway. ConclusionOur study elucidates the functional role of METTL1 in AM's invasive development and prognosis. High METTL1 expression is linked to postoperative recurrence, and METTL1 appears to promote AM invasiveness through the MAPK signaling pathway. These findings contribute to a better understanding of AM pathogenesis and may guide future therapeutic strategies.

Abstract TP283: Histone Lactylation and Neuronal Autophagy: Insights Into the Penumbra-Core Transition and Potential Biomarker in Ischemic Stroke

Objectives: The dynamic evolution of ischemic penumbra to core has implications for stroke outcomes. Optimizing outcomes remains challenging despite advanced recanalization. We examined the intricate molecular interplay between lactylation and RNA m6A modifications, to unravel the H3K18la-Tfeb m6A-autophagy-neuron-injury pathway, understanding mechanisms underlying penumbra-core transition and investigating potential biomarkers indicative of injury severity. Method: Adult SD rats underwent 2, 6, and 24 h of middle cerebral artery occlusion (MCAO) and subsequent reperfusion. The penumbra-core transition-induced brain damage was quantified by infarct volume post 48 h of reperfusion. Employing CUT&Tag and ChIP, H3K18la enrichment in promoter regions was identified. The mRNA and protein levels of lactylation, methyltransferase like 3 (METTL3), transcription factor EB (TFEB) and autophagy-related molecules (LC3, P62, LAMP1) were examined. METTL3 and TFEB localization was determined using immunofluorescence, while serum H3K18la levels were analyzed. Result: Ischemia duration was directly linked to elevated neuronal lactylation, particularly H3K18la, and autophagy. Increased H3K18la enrichment was observed in Mettl3 promoter regions, promoting Mettl3 gene transcription. This leads to METTL3-induced tfeb m6A modifications, enhancing TFEB expression and its nuclear translocation. Administering C646 (lactylation writer inhibitor) suppressed METTL3, TFEB, LC3, P62, and LAMP1 expressions, which mitigated brain injuries, underlining lactylation's key role in excessive neuronal autophagy initiation. All molecular alterations were strongly tied to the penumbra-core transition. Markedly, ischemic rats presented elevated serum H3K18la levels, which correlated with dynamic neurological damage. Conclusion: Our study elucidates molecular dynamics driving the penumbra-core transition in ischemic stroke. H3K18la initiatives METTL3-associated tfeb m6A modifications, activating the autophagy-lysosome pathway, thus enhancing autophagy and neuronal death. Serum H3K18la variations emerge as potential indicators of neuronal damage severity, suggesting effective biomarkers for assessing post-stroke prognosis.

Methyltransferase METTL3-mediated maturation of miR-4654 facilitates high glucose-induced apoptosis and oxidative stress in lens epithelial cells via decreasing SOD2.

N6-methyladenosine (m6 A) modification has been reported to have roles in modulating the development of diabetic cataract (DC). Methyltransferase-like 3 (METTL3) is a critical m6 A methyltransferase involving in m6 A modification activation. Here, we aimed to explore the action and mechanism of METTL3-mediated maturation of miR-4654 in DC progression. Human lens epithelial cells (HLECs) were exposed to high glucose (HG) to imitate DC condition invitro. Levels of genes and proteins were tested via qRT-PCR and western blotting assays. The proliferation and apoptosis of HLECs were evaluated by cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays, respectively. Oxidative stress was analyzed by detecting the contents of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA). The binding of miR-4654 and SOD2 was confirmed by dual-luciferase reporter assay. The m6 A-RNA immunoprecipitation (MeRIP) assay detected the m6 A modification profile. Thereafter, we found that miR-4654 expression was elevated in DC samples and HG-induced HLECs. MiR-4654 knockdown reversed HG-mediated apoptosis and oxidative stress in HLECs. Mechanistically, miR-4654 directly targeted SOD2, silencing of SOD2 abolished the protective effects of miR-4654 knockdown on HLECs under HG condition. In addition, METTL3 induced miR-4654 maturation through promoting pri-miR-4654 m6 A modification, thereby increasing miR-4654 content in HLECs. METTL3 was highly expressed in DC samples and HG-induced HLECs, METTL3 deficiency protected HLECs against HG-mediated apoptotic and oxidative injury via down-regulating miR-4654. In all, METTL3 induced miR-4654 maturation in a m6 A-dependent manner, which was then reduced SOD2 expression, thus promoting apoptosis and oxidative stress in HLECs, suggesting a novel path for DC therapy.

Methyltransferase-like 3 mediated RNA m6 A modifications in the reproductive system: Potentials for diagnosis and therapy.

Several studies have highlighted the functional indispensability of methyltransferase-like 3 (METTL3) in the reproductive system. However, a review that comprehensively interprets these studies and elucidates their relationships is lacking. Therefore, the present work aimed to review studies that have investigated the functions of METTL3 in the reproductive system (including spermatogenesis, follicle development, gametogenesis, reproductive cancer, asthenozoospermia and assisted reproduction failure). This review suggests that METTL3 functions not only essential for normal development, but also detrimental in the occurrence of disorders. In addition, promising applications of METTL3 as a diagnostic or prognostic biomarker and therapeutic target for reproductive disorders have been proposed. Collectively, this review provides comprehensive interpretations, novel insights, potential applications and future perspectives on the role of METTL3 in regulating the reproductive system, which may be a valuable reference for researchers and clinicians.

METTL3-mediated m6A modification of EPPK1 to promote the development of esophageal cancer through regulating the PI3K/AKT pathway.

Methyltransferase like 3 (METTL3) has been proved to be involved in the progression of various cancers. In this study, we explored the role of METTL3 and its underlying mechanism in esophageal cancer progression. The mRNA and protein levels of METTL3 and epiplakin1 (EPPK1) were determined using qRT-PCR and western blot. The proliferative ability was evaluated through 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT), colony formation, and EdU assays. Transwell invasion assay and wound-healing assay were employed for detecting cell invasion and migration, respectively. Cell stemness was evaluated by sphere-formation assay. Xenograft tumor experiments and immunohistochemistry (IHC) were performed to explore the effects of METTL3 knockdown on tumor growth in vivo. The N6-methyladenosine (m6A) modification of EPPK1 was analyzed using MeRIP. RNA-protein immunoprecipitation (RIP) and dual-luciferase reporter assays were used to verify the relationship between EPPK1 and METTL3. METTL3 was upregulated in esophageal cancer tissues and cells, which was related to the poor prognosis of esophageal cancer patients. Knockdown of METTL3 overtly decreased the proliferative, invasive, migrated abilities, and cell stemness of esophageal cancer cells in vitro. Moreover, depletion of METTL3 also observably suppressed the growth of tumor in vivo. EPPK1 was a direct target of METTL3, and METTL3 could mediate the m6A modification of EPPK1. EPPK1 was downregulated in esophageal cancer tissues and cells, and EPPK1 depletion markedly repressed cell proliferation, invasion, migration, and stemness of esophageal cancer cells. The inhibition effects of METTL3 deficiency on these malignant behaviors were harbored by EPPK1 upregulation in esophageal cancer cells. In addition, METTL3 deficiency reduced EPPK1 expression to inactivate the PI3K/AKT pathway. Our results revealed that METTL3 deficiency regulated the m6A modification of EPPK1 to inhibit the PI3K/AKT pathway, thereby restraining the progression of esophageal cancer.

Follicle-stimulating hormone accelerates osteoclast migration by enhancing methyltransferase-like 3-mediated m6A methylation of cathepsin K.

Follicle-stimulating hormone (FSH) accelerates osteoporosis in postmenopausal women, while the underlying mechanism remains uncharacterized. N6-methyladenosine (m6A) is one of the most important regulations in the development of osteoporosis. In this study, we aimed to investigate the role of FSH in m6A modification and osteoclast function. Here, we showed that FSH upregulated m6A levels in osteoclasts via stimulating methyltransferase-like 3 (METTL3) protein expression. FSH enhanced osteoclast migration, while the knockdown of METTL3 eliminated this enhancement. Both MeRIP-seq and RNA sequencing identified that cathepsin K (CTSK) is the potential downstream target of METTL3. Knockdown of CTSK reduced FSH-upregulated osteoclast migration. Furthermore, silencing METTL3 decreased CTSK mRNA stability. Finally, FSH induced phosphorylation of cyclic-AMP response element-binding protein (CREB), while silencing of CREB attenuated the effects of FSH on the promoter transcriptional activity of Mettl3 and CTSK/METTL3 protein. Taken together, these findings indicate that FSH promotes osteoclast migration via the CREB/METTL3/CTSK signaling pathway, which may provide a potential target for suppressing osteoclast mobility and postmenopausal osteoporosis therapy.

The RNA m6A writer METTL3 in tumor microenvironment: emerging roles and therapeutic implications.

The tumor microenvironment (TME) is a heterogeneous ecosystem comprising cancer cells, immune cells, stromal cells, and various non-cellular components, all of which play critical roles in controlling tumor progression and response to immunotherapies. Methyltransferase-like 3 (METTL3), the core component of N 6-methyladenosine (m6A) writer, is frequently associated with abnormalities in the m6A epitranscriptome in different cancer types, impacting both cancer cells and the surrounding TME. While the impact of METTL3 on cancer cells has been extensively reviewed, its roles in TME and anti-cancer immunity have not been comprehensively summarized. This review aims to systematically summarize the functions of METTL3 in TME, particularly its effects on tumor-infiltrating immune cells. We also elaborate on the underlying m6A-dependent mechanism. Additionally, we discuss ongoing endeavors towards developing METTL3 inhibitors, as well as the potential of targeting METTL3 to bolster the efficacy of immunotherapy.

METTL3 Modulates TXNIP Expression to Affect the Activation of NLRP3 Inflammasome in Hepatic Cells Under Oxygen-Glucose Deprivation/Reperfusion Injury.

Hepatic ischemia-reperfusion (I/R) injury is still a major risk factor and unsolved problem in hepatic surgery. Methyltransferase-like 3 (METTL3), an important m6A-modified methylase, regulates inflammation and cellular stress response. In this study, we demonstrated the special role of METTL3 and its underlying mechanism in hepatic I/R injury. In the mouse model of hepatic I/R and in the oxygen-glucose deprivation and reoxygenation (OGD/R)-induced AML12 and NCTC 1469 cells, the expression of METTL3 was significantly upregulated. Inhibition of METTL3 in OGD/R-induced AML12 and NCTC 1469 cells both increased the cell viability, declined the cell apoptosis, and decreased the reactive oxygen species (ROS) and the release levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18), diminishing NLRP3 and Caspase1-p20 expressions. Moreover, METTL3 positively modulated TXNIP expression in an m6A manner. TXNIP overexpression reversed the effects of METTL3 knockdown on OGD/R-induced injury in AML12 cells. Furthermore, inhibition of NLRP3 inflammasome activity contributed to the protective effects of TXNIP knockdown in OGD/R-induced AML12 cells. In conclusion, METTL3 knockdown alleviated OGD/R-induced hepatocyte injury, and the specific mechanism was associated with the inhibition of NLRP3 inflammasome activation, which was attributed to the reduction of TXNIP in an m6A-dependent manner.

METTL14 depletion ameliorates ferroptosis in severe acute pancreatitis by increasing the N6-methyladenosine modification of ACSL4 and STA1

Methyltransferase-like 14 (METTL14) is implicated in the regulation of various inflammatory disorders. However, its function and molecular mechanism in severe acute pancreatitis (SAP) remains unrevealed. Here we reported an increase in METTL14 in the pancreas of SAP mice and cerulein-LPS-treated AR42J cells. METTL14 depletion reversed inflammatory response and ferroptosis by reducing the expression of SAT1 (spermidine/spermine N1-acetyltransferase 1) and ACSL4 (acyl-CoA synthetase long chain family member 4) in an m6A-dependent manner. IGF2BP2 (insulin like growth factor 2 mRNA binding protein 2) could recognize m6A-modified SAT1 and ACSL4 mRNA and enhance their stability. Moreover, METTL14 depletion ameliorated pancreatic injury, inflammation, and ferroptosis induced by SAP. METTL14 overexpression aggravated SAP by promoting ferroptosis in vivo. Therefore, these results demonstrated that METTL14-induced ferroptosis promoted the progression of SAP, and targeting METTL14 or ferroptosis could be a potential strategy for the prevention and treatment of SAP.

Downregulation of Wtap causes dilated cardiomyopathy and heart failure

RNA binding proteins have been shown to regulate heart development and cardiac diseases. However, the detailed molecular mechanisms is not known. In this study, we identified Wilms' tumor 1-associating protein (WTAP, a key regulatory protein of the m6A RNA methyltransferase complex) as a key regulator of heart function and cardiac diseases. WTAP is associated with heart development, and its expression is downregulated in both human and mice with heart failure. Cardiomyocyte-specific knockout of Wtap (Wtap-CKO) induces dilated cardiomyopathy, heart failure and neonatal death. Although WTAP deficiency in the heart decreases METTL3 (methyltransferase-like 3) protein levels, cardiomyocyte-specific overexpression of Mettl3 in Wtap-CKO mice does not rescue the phenotypes of Wtap-CKO mice. Instead, WTAP deficiency in the heart decreases chromatin accessibility in the promoter regions of Mef2a (myocyte enhancer factor-2α) and Mef2c, leading to reduced mRNA and protein levels of these genes and lower expression of their target genes. Conversely, WTAP directly binds to the promoter of the Mef2c gene and increases its promoter luciferase activity and expression. These data demonstrate that WTAP plays a key role in heart development and cardiac function by maintaining the chromatin accessibility of cardiomyocyte specific genes.

METTL14 contributes to acute lung injury by stabilizing NLRP3 expression in an IGF2BP2-dependent manner

Acute lung injury (ALI) as well as its more severe form, acute respiratory distress syndrome (ARDS), frequently leads to an uncontrolled inflammatory response. N6-methyladenosine (m6A) modification was associated with the progression of several inflammatory diseases. However, the role of methyltransferase-like 14 (METTL14)-mediated m6A methylation in ALI/ARDS remains unclear. Here, we reported an increase in overall expression levels of m6A and METTL14 in circulating monocyte-derived macrophages recruited to the lung following ALI, which is correlated with the severity of lung injury. We further demonstrated the critical function of METTL14 in activating NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome in vitro and in mouse models of ALI/ARDS, and validated NLRP3 as the downstream target of METTL14 by the m6A RNA immunoprecipitation (MeRIP) and RIP assays. Mechanistically, METTL14-methylated NLRP3 transcripts were subsequently recognized by insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), an m6A reader, which stabilized NLRP3 mRNA. Furthermore, we observed that IGF2BP2 knockdown diminished LPS-induced ALI in mice by downregulating NLRP3 expression. In summation, our study revealed that the molecular mechanism underlying the pathogenesis of ALI/ARDS involves METTL14-mediated activation of NLRP3 inflammasome in an IGF2BP2 dependent manner, thereby demonstrating the potential of METTL14 and IGF2BP2 as promising biomarkers and therapeutic targets for ALI/ARDS treatment.

METTL3 attenuates ferroptosis sensitivity in lung cancer via modulating TFRC.

Overexpression of methyltransferase-like 3 (METTL3) is significantly correlated with the malignancy of lung cancer (LC). In the present study, we demonstrated that METTL3 had higher levels in LC tissues relative to normal tissues. METTL3 showed superior sensitivity and specificity for diagnosis and identification of LC functions. In addition, silencing METTL3 resulted in enhanced ferroptosis sensitivity, whereas overexpression of METTL3 exhibited the opposite effect. Inhibition of METTL3 impeded LC growth in cell-derived xenografts. Further exploratory studies found that METTL3 stimulated the low expression of transferrin receptor (TFRC), which was critical for ferroptosis sensitization in LC cells induced by silenced METTL3, as silencing of TFRC caused a decrease in negative regulators of ferroptosis (FTH1 and FTL) in METTL3 knockdown A549 and PC9 cells. Finally, we confirmed that METTL3 attenuation effectively maintained the stability of TFRC mRNA. In conclusion, we reported a novel mechanism of METTL3 desensitization to ferroptosis via regulating TFRC, and an appropriate reduction of METTL3 might sensitize cancer cells to ferroptosis-based therapy.

FOXM1 Participates in Scleral Remodeling in Myopia by Upregulating APOA1 Expression Through METTL3/YTHDF2.

Apolipoprotein A1 (APOA1) is a potential crucial protein and treatment goal for pathological myopia in humans. This study set out to discover the function of APOA1 in scleral remodeling in myopia and its underlying mechanisms. A myopic cell model was induced using hypoxia. Following loss- and gain-of function experiments, the expression of the myofibroblast transdifferentiation-related and collagen production-related factors Forkhead box M1 (FOXM1), APOA1, and methyltransferase-like 3 (METTL3) in the myopic cell model was examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The proliferation and apoptosis were determined by Cell Counting Kit-8 assay and flow cytometry, respectively. Chromatin immunoprecipitation (ChIP) was employed to examine FOXM1 enrichment in the METTL3 promoter, methylated RNA immunoprecipitation (Me-RIP) to examine the N6-methyladenosine (m6A) modification level of APOA1, and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to examine the binding between METTL3 and APOA1. Hypoxia-induced human scleral fibroblasts (HSFs) had high APOA1 and FOXM1 expression and low METTL3 expression. FOXM1 knockdown elevated METTL3 expression and downregulated APOA1 expression. FOXM1 was enriched in METTL3 promoter. APOA1 or FOXM1 knockdown or METTL3 overexpression reversed the hypoxia-induced elevation in vinculin, paxillin, and α-smooth muscle actin (α-SMA) levels and apoptosis and the reduction in collagen, type I, alpha 1 (COL1A1) level and cell proliferation in HSFs. METTL3 or YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) knockdown or APOA1 overexpression reversed the impacts of FOXM1 knockdown on vinculin, paxillin, α-SMA, and COL1A1 expression and cell proliferation and apoptosis. FOXM1 elevated the m6A methylation level of APOA1 by repressing METTL3 transcription and enhanced APOA1 mRNA stability and transcription by reducing the YTHDF2-recognized m6A methylated transcripts.

METTL3-mediated m6A modification of SOX4 regulates osteoblast proliferation and differentiation via YTHDF3 recognition

N6-methyladenosine (m6A), the most prevalent internal modification in mRNA, is related to the pathogenesis of osteoporosis (OP). Although methyltransferase Like-3 (METTL3), an m6A transferase, has been shown to mitigate OP progression, the mechanisms of METTL3-mediated m6A modification in osteoblast function remain unclear. Here, fluid shear stress (FSS) induced osteoblast proliferation and differentiation, resulting in elevated levels of METTL3 expression and m6A modification. Through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and Transcriptomic RNA Sequencing (RNA-seq), SRY (Sex Determining Region Y)-box 4 (SOX4) was screened as a target of METTL3, whose m6A-modified coding sequence (CDS) regions exhibited binding affinity towards METTL3. Further functional experiments demonstrated that knockdown of METTL3 and SOX4 hampered osteogenesis, and METTL3 knockdown compromised SOX4 mRNA stability. Via RNA immunoprecipitation (RIP) assays, we further confirmed the direct interaction between METTL3 and SOX4. YTH N6-Methyladenosine RNA Binding Protein 3 (YTHDF3) was identified as the m6A reader responsible for modulating SOX4 mRNA and protein levels by affecting its degradation. Furthermore, in vivo experiments demonstrated that bone loss in an ovariectomized (OVX) mouse model was reversed through the overexpression of SOX4 mediated by adeno-associated virus serotype 2 (AAV2). In conclusion, our research demonstrates that METTL3-mediated m6A modification of SOX4 plays a crucial role in regulating osteoblast proliferation and differentiation through its recognition by YTHDF3. Our research confirms METTL3-m6A-SOX4-YTHDF3 as an essential axis and potential mechanism in OP.

Methyltransferase-like 3 (METTL3) Epigenetically Modulates Glutathione Peroxidase 4 (GPX4) Expression to Affect Asthma.

Asthma, a prevalent chronic airway inflammatory condition, poses a significant health challenge. In this study, we delved into the regulatory mechanisms governing asthma, focusing on Methyltransferase-like 3 (METTL3). Through an ovalbumin (OVA)-induced mouse model and interleukin-13 (IL-13)-induced cell model, we mimicked the in vivo and in vitro functions of METTL3 in asthma. Our research revealed that METTL3 expression significantly decreased in asthma-induced mice and IL-13-stimulated cells compared to the control group. Moreover, METTL3 overexpression enhanced bronchial epithelial cell viability and proliferation. Mechanistically, we observed elevated levels of total iron, Fe2+, malondialdehyde (MDA), lipid reactive oxygen species (ROS), alongside reduced glutathione (GSH) levels in IL-13-stimulated cells. Remarkably, METTL3 overexpression counteracted these effects, suggesting a pivotal role in mitigating asthma-related oxidative stress. Furthermore, our study highlighted the involvement of N6-methyladenosine methylation (m6A) modification, where METTL3 regulated the m6A modification of glutathione peroxidase 4 (GPX4) RNA, impacting RNA stability. Knockdown of METTL3 suppressed m6A modification on GPX4 RNA, impairing its stability and contributing to IL-13-induced ferroptosis. Interestingly, METTL3 overexpression not only inhibited cell ferroptosis but also alleviated asthma symptoms. Our findings shed light on the epigenetic regulation of asthma through METTL3-mediated m6A modification, offering potential therapeutic avenues for this prevalent inflammatory disease.

N6-methyladenosine-induced miR-182-5p promotes multiple myeloma tumorigenesis by regulating CAMK2N1.

Methyltransferase like 3 (METTL3) has been reported to promote tumorigenesis of multiple myeloma (MM), however, the molecular mechanism still needs further research. The N6-methyladenosine (m6A) level in tissues or cells was measured by m6A kit and dot blot assay. The mRNA and protein expression were detected by quantitative real-time PCR (RT-qPCR) and Western blot, respectively. The cell counting kit-8 and colony formation assay were used to detect the cell proliferation. Coimmunoprecipitation (Co-IP) experiment verified the binding of two proteins. The luciferase reporter experiment demonstrated the targeted binding of miR-182-5p and CaMKII inhibitor 1 (CAMK2N1). More importantly, tumor growth was measured in xenograft mice. Our data showed that the expression of METTL3 was significantly increased in MM patients' samples and MM cells. METTL3 overexpression promoted MM cells proliferation, and METTL3 knockdown inhibited MM cells proliferation. Mechanically, METTL3-dependent m6A participated in DiGeorge syndrome critical region 8 (DGCR8)-mediated maturation of pri-miR-182. Upregulation of miR-182-5p further enhanced the promoting proliferation effect of METTL3 overexpression on MM cells. Moreover, the luciferase reporter gene experiment proved that miR-182-5p targetedly regulated CAMK2N1 expression. Xenograft tumor in nude mice further verified that METTL3 promoted MM tumor growth through miR-182/CAMK2N1 signal axis. In summary, the METTL3/miR-182-5p/CAMK2N1 axis plays an important role in MM tumorigenesis, which may provide a new target for MM therapy.

Study on the Role and Mechanism of METTL3 Mediating the Up-regulation of m6A Modified Long Non-coding RNA THAP7-AS1 in Promoting the Occurrence of Lung Cancer

Lung cancer is a major threat to human health. The molecular mechanisms related to the occurrence and development of lung cancer are complex and poorly known. Exploring molecular markers related to the development of lung cancer is helpful to improve the effect of early diagnosis and treatment. Long non-coding RNA (lncRNA) THAP7-AS1 is known to be highly expressed in gastric cancer, but has been less studied in other cancers. The aim of the study is to explore the role and mechanism of methyltransferase-like 3 (METTL3) mediated up-regulation of N6-methyladenosine (m6A) modified lncRNA THAP7-AS1 expression in promoting the development of lung cancer. Samples of 120 lung cancer and corresponding paracancerous tissues were collected. LncRNA microarrays were used to analyze differentially expressed lncRNAs. THAP7-AS1 levels were detected in lung cancer, adjacent normal tissues and lung cancer cell lines by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The diagnostic value of THAP7-AS1 in lung cancer and the relationship between THAP7-AS1 expression and survival rate and clinicopathological parameters were analyzed. Bioinformatics analysis, methylated RNA immunoprecipitation (meRIP), RNA pull-down and RNA-immunoprecipitation (RIP) assay were used to investigate the molecular regulation mechanism of THAP7-AS1. Cell proliferation, migration, invasion and tumorigenesis of SPC-A-1 and NCI-H1299 cells were determined by MTS, colony-formation, scratch, Transwell and xenotransplantation in vivo, respectively. Expression levels of phosphoinositide 3-kinase/protein kenase B (PI3K/AKT) signal pathway related protein were detected by Western blot. Expression levels of THAP7-AS1 were higher in lung cancer tissues and cell lines (P<0.05). THAP7-AS1 has certain diagnostic value in lung cancer [area under the curve (AUC)=0.737], and its expression associated with overall survival rate, tumor size, tumor-node-metastasis (TNM) stage and lymph node metastasis (P<0.05). METTL3-mediated m6A modification enhanced THAP7-AS1 expression. The cell proliferation, migration, invasion and the volume and mass of transplanted tumor were all higher in the THAP7-AS1 group compared with the NC group and sh-NC group of SPC-A-1 and NCI-H1299 cells, while the cell proliferation, migration and invasion were lower in the sh-THAP7-AS1 group (P<0.05). THAP7-AS1 binds specifically to Cullin 4B (CUL4B). The cell proliferation, migration, invasion, and expression levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphoinositide-3 kinase, catalytic subunit delta (PIK3CD), phospho-phosphatidylinositol 3-kinase (p-PI3K), phospho-protein kinase B (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) were higher in the THAP7-AS1 group compared with the Vector group of SPC-A-1 and NCI-H1299 cells (P<0.05). LncRNA THAP7-AS1 is stably expressed through m6A modification mediated by METTL3, and combines with CUL4B to activate PI3K/AKT signal pathway, which promotes the occurrence and development of lung cancer.