Knockdown Of Methyltransferase-like 3 Research Articles

METTL14 contributes to the progression of nasopharyngeal carcinoma through regulating the stability of AOC1 mRNA

BackgroundNasopharyngeal carcinoma (NPC) is a malignant epithelial tumor of the nasopharyngeal mucosa with a high incidence rate all over the world. Methyltransferase-like 14 (METTL14) is a major RNA N6-adenosine methyltransferase implicated in tumor progression by regulating RNA function. This study is designed to explore the biological function and mechanism of METTL14 in NPC.MethodsMETTL14 and Amine oxidase copper containing 1 (AOC1) expression were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The protein levels of METTL14, AOC1, Cyclin D1, B-cell lymphoma-2 (Bcl-2), and N-cadherin were measured using western blot. Cell proliferation, cycle progression, apoptosis, migration, and invasion were assessed using 5-ethynyl-2’-deoxyuridine (EdU), Colony formation, flow cytometry, wound scratch, and transwell assays. The interaction between METTL14 and AOC1 was verified using RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), and dual-luciferase reporter assays. The biological role of METTL14 on NPC tumor growth was examined by the xenograft tumor model in vivo.ResultsMETTL14 and AOC1 were highly expressed in NPC tissues and cells. Moreover, METTL14 knockdown might block NPC cell proliferation, migration, invasion, and induce cell apoptosis in vitro. In mechanism, METTL14 might enhance the stability of AOC1 mRNA via m6A methylation. METTL14 silencing might repress NPC tumor growth in vivo.ConclusionMETTL14 might boosted the development of NPC cells partly by regulating the stability of AOC1 mRNA, which provided a promising therapeutic target for NPC treatment.

Methyltransferase like-14 suppresses growth and metastasis of non-small-cell lung cancer by decreasing LINC02747.

Multiple epigenetic regulatory mechanisms exert critical roles in tumor development, and understanding the interactions and impact of diverse epigenetic modifications on gene expression in cancer is crucial for the development of precision medicine. We found that methyltransferase-like 14 (METTL14) was significantly downregulated in non-small-cell lung cancer (NSCLC) tissues. Functional experiments demonstrated that overexpression of METTL14 inhibited the proliferation and migration of NSCLC cells both invivo and invitro, and the colorimetric m6A quantification assay also showed that knockdown of METTL14 notably reduced global m6A modification levels in NSCLC cells. By using the methylated-RNA immunoprecipitation-qPCR and dual-luciferase reporter assays, we verified that long noncoding RNA LINC02747 was a target of METTL14 and was regulated by METTL14-mediated m6A modification, and silencing LINC02747 inhibited the malignant progression of NSCLC by modulating the PI3K/Akt and CDK4/Cyclin D1 signaling pathway. Further studies revealed that overexpression of METTL14 promoted m6A methylation and accelerated the decay of LINC02747 mRNA via increased recognition of the "GAACU" binding site by YTHDC2. Additionally, histone demethylase lysine-specific histone demethylase 5B (KDM5B) mediated the demethylation of histone H3 lysine 4 tri-methylation (H3K4me3) in the METTL14 promoter region and repressed its transcription. In summary, KDM5B downregulated METTL14 expression at the transcriptional level in a H3K4me3-dependent manner, while METTL14 modulated LINC02747 expression via m6A modification. Our results demonstrate a synergy of multiple mechanisms in regulating the malignant phenotype of NSCLC, revealing the complex regulation involved in the occurrence and development of cancer.

METTL14 regulates CD8+T-cell activation and immune responses to anti‐PD‐1 therapy in lung cancer

BackgroundN6-methyladenosine (m6A) modification plays an important role in lung cancer. However, methyltransferase-like 14 (METTL14), which serves as the main component of the m6A complex, has been less reported to be involved in the immune microenvironment of lung cancer. This study aimed to analyze the relationship between METTL14 and the immune checkpoint inhibitor programmed death receptor 1 (PD-1) in lung cancer.MethodsCCK-8, colony formation, transwell, wound healing, and flow cytometry assays were performed to explore the role of METTL14 in lung cancer progression in vitro. Furthermore, syngeneic model mice were treated with sh-METTL14 andan anti-PD-1 antibody to observe the effect of METTL14 on immunotherapy. Flow cytometry and immunohistochemical (IHC) staining were used to detect CD8 expression. RIP and MeRIP were performed to assess the relationship between METTL14 and HSD17B6. LLC cells and activated mouse PBMCs were cocultured in vitro to mimic immune cell infiltration in the tumor microenvironment. ELISA was used to detect IFN-γ and TNF-α levels.ResultsThe online database GEPIA showed that high METTL14 expression indicated a poor prognosis in patients with lung cancer. In vitro assays suggested that METTL14 knockdown suppressed lung cancer progression. In vivo assays revealed that METTL14 knockdown inhibited tumor growth and enhanced the response to PD-1 immunotherapy. Furthermore, METTL14 knockdown enhanced CD8+T-cell activation and infiltration. More importantly, METTL14 knockdown increased the stability of HSD17B6 mRNA by reducing its m6A methylation. In addition, HSD17B6 overexpression promoted the activation of CD8+ T cells.ConclusionThe disruption of METTL14 contributed to CD8+T-cell activation and the immunotherapy response to PD-1 via m6A modification of HSD17B6, thereby suppressing lung cancer progression.

METTL14-mediated N6-methyladenosine modification induces the ferroptosis of hypoxia/reoxygenation-induced cardiomyocytes

BackgroundHypoxia/reoxygenation (H/R) induces cardiomyocyte ferroptosis, a core remodeling event in myocardial ischemia/reperfusion injury. Methyltransferase-like 14 (METTL14) emerges as a writer of N6-methyladenosine (m6A) modification. This study was conducted to decipher the role of METTL14 in H/R-induced cardiomyocyte ferroptosis.MethodsMouse cardiomyocytes HL-1 were cultured and underwent H/R treatment. The degree of ferroptosis after H/R treatment was appraised by the cell counting kit-8 assay, assay kits (ROS/GSH/Fe2+), and Western blotting (GPX4/ACSL4). The intracellular expressions of METTL14, pri-miR-146a-5p, miR-146a-5p, or adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) were examined by real-time quantitative polymerase chain reaction or Western blotting, with m6A quantification analysis and RNA immunoprecipitation to determine the total m6A level and the expression of pri-miR-146a-5p bound to DiGeorge critical region 8 (DGCR8) and m6A-modified pri-miR-146a-5p. The binding of miR-146a-5p to APPL1 was testified by the dual-luciferase assay.ResultsH/R treatment induced cardiomyocyte ferroptosis (increased ROS, Fe2+, and ACSL4 and decreased GSH and GPX4) and upregulated METTL14 expression. METTL14 knockdown attenuated H/R-induced cardiomyocyte ferroptosis. METTL14 induced the recognition of pri-miR-146a-5p by DGCR8 by increasing m6A modification on pri-miR-146a-5p, which promoted the conversion of pri-miR-146a-5p into miR-146a-5p and further repressed APPL1 transcription. miR-146a-5p upregulation or APPL1 downregulation limited the inhibitory effect of METTL14 downregulation on H/R-induced cardiomyocyte ferroptosis.ConclusionMETTL14 promoted miR-146a-5p expression through the recognition and processing of pri-miR-146a-5p by DGCR8, which repressed APPL1 transcription and triggered H/R-induced cardiomyocyte ferroptosis.

METTL14 promotes neuroblastoma formation by inhibiting YWHAH via an m6A-YTHDF1-dependent mechanism

Neuroblastoma (NB) is a common childhood tumor with a high incidence worldwide. The regulatory role of RNA N6-methyladenosine (m6A) in gene expression has attracted significant attention, and the impact of methyltransferase-like 14 (METTL14) on tumor progression has been extensively studied in various types of cancer. However, the specific influence of METTL14 on NB remains unexplored. Using data from the Target database, our study revealed significant upregulation of METTL14 expression in high-risk NB patients, with strong correlation with poor prognosis. Furthermore, we identified ETS1 and YY1 as upstream regulators that control the expression of METTL14. In vitro experiments involving the knockdown of METTL14 in NB cells demonstrated significant inhibition of cell proliferation, migration, and invasion. In addition, suppressing METTL14 inhibited NB tumorigenesis in nude mouse models. Through MeRIP-seq and RNA-seq analyses, we further discovered that YWHAH is a downstream target gene of METTL14. Mechanistically, we observed that methylated YWHAH transcripts, particularly those in the 5′ UTR, were specifically recognized by the m6A “reader” protein YTHDF1, leading to the degradation of YWHAH mRNA. Moreover, the downregulation of YWHAH expression activated the PI3K/AKT signaling pathway, promoting NB cell activity. Overall, our study provides valuable insights into the oncogenic effects of METTL14 in NB cells, highlighting its role in inhibiting YWHAH expression through an m6A-YTHDF1-dependent mechanism. These findings also suggest the potential utility of a biomarker panel for prognostic prediction in NB patients.

METTL3 promotes trophoblast ferroptosis in preeclampsia by stabilizing the ACSL4 m6A modification

This study aims to explore the role of methyltransferase-like 3 (METTL3) modulation of ferroptosis in the pathogenesis of trophoblast-mediated preeclampsia. The expression of METTL3 and acyl-CoA synthetase long chain family member 4 (ACSL4) was measured in clinical placental tissues and trophoblasts using qPCR and Western blot techniques. The effects of METTL3 on the symptoms of preeclampsia were also validated in rat models. METTL3 and ACSL4 were upregulated in placental tissues from patients with preeclampsia and in hypoxia-induced trophoblasts. METTL3 silencing increased the migration and invasion of trophoblasts cultured under hypoxic conditions. Knockdown of METTL3 increased cell viability and suppressed ferroptosis in hypoxia-stimulated trophoblasts. Hypoxia increased the level of m6A in cells, whereas silencing METTL3 partially reversed this change. Silencing METTL3 resulted in a decrease in m6A modification of ACSL4 mRNA, which led to a reduction in ACSL4 mRNA stability. ACSL4 upregulation partially reversed the effects of METTL3 silencing on cell viability, migration, invasion, and ferroptosis in hypoxia-stimulated trophoblasts. Inhibition of METTL3 in preeclampsia rats decreased blood pressure, urine protein levels, fetal survival rate, and ACSL4-mediated ferroptosis. METTL3 elevates ferroptosis to inhibit the migration and invasion of trophoblasts and in vivo preeclampsia symptoms by catalyzing the m6A modification of ACSL4 mRNA.

METTL3 knockdown suppresses RA-FLS activation through m6A-YTHDC2-mediated regulation of AMIGO2

The dysregulation of N6-methyladenosine (m6A) on mRNAs is involved in the pathogenesis of rheumatoid arthritis (RA). Methyltransferase-like 3 (METTL3), serving as a central m6A methyltransferase, is highly expressed in macrophages, synovial tissues and RA fibroblast-like synoviocytes (RA-FLS) of RA patients. However, METTL3-mediated m6A modification on target mRNAs and the molecular mechanisms involved in RA-FLS remain poorly defined. Our research demonstrated that METTL3 knockdown decreased the proliferation, migratory and invasive abilities of RA-FLS. Notably, we identified the adhesion molecule with Ig like domain 2 (AMIGO2) as a probable downstream target of both METTL3 and YTH Domain Containing 2 (YTHDC2) in RA-FLS. We revealed that AMIGO2 augmented the activation of RA-FLS and can potentially reverse the phenotypic effects induced by the knockdown of either METTL3 or YTHDC2. Mechanistically, METTL3 knockdown decreased m6A modification in the 5′-untranslated region (5′UTR) of AMIGO2 mRNA, which diminished its interaction with YTHDC2 in RA-FLS. Our findings unveiled that silencing of METTL3 inhibited the proliferation and aggressive behaviors of RA-FLS by downregulating AMIGO2 expression in an m6A-YTHDC2 dependent mechanism, thereby underscoring the pivotal role of the METTL3-m6A-YTHDC2-AMIGO2 axis in modulating RA-FLS phenotypes.

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 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.

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.

Investigation of METTL3 as an inhibitor of kanamycin-induced ototoxicity via stress granule formation.

Methyltransferase-like 3 (METTL3), a component of the N6-methyladenosine (m6A) methyltransferase family, exhibits significant expression in HEI-OC1 cells and cochlear explants. Aminoglycoside antibiotics, known for their ototoxic potential, frequently induce irreversible auditory damage in hair cells, predominantly through oxidative stress mechanisms. However, the specific role of METTL3 in kanamycin-induced hair cell loss remains unclear. This study aims to elucidate the mechanisms by which METTL3 contributes to kanamycin-induced ototoxicity. In vivo experiments demonstrated a notable reduction in METTL3 expression within cochlear explants following kanamycin administration, concomitant with the formation of stress granules (SGs). Similarly, a 24-hour kanamycin treatment led to decreased METTL3 expression and induced SG formation both in HEI-OC1 cells and neonatal cochlear explants, corroborating the in vivo observations. Lentivirus-mediated transfection was employed to overexpress and knockdown METTL3 in HEI-OC1 cells. Knockdown of METTL3 resulted in increased reactive oxygen species (ROS) levels and apoptosis induced by kanamycin, while concurrently reducing SG formation. Conversely, overexpression of METTL3 attenuated ROS generation, decreased apoptosis rates, and promoted SG formation induced by kanamycin. Therefore, METTL3-mediated SG formation presents a promising target for mitigating kanamycin-induced ROS generation and the rate of apoptosis. This finding indicates that METTL3-mediated SG formation holds potential in mitigating kanamycin-induced impairments in cochlear hair cells by reducing ROS formation and apoptosis rates.

Inhibition of METTL3 alleviated LPS-induced alveolar epithelial cell apoptosis and acute lung injury via restoring neprilysin expression

AimsTo investigate the role and mechanisms of methyltransferase-like 3 (METTL3) in the pathogenesis of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Main methodsLPS intratracheally instillation was applied in alveolar epithelial cell METTL3 conditional knockout (METTL3-CKO) mice and their wild-type littermates. In addition, METTL3 inhibitor STM2457 was used. LPS treatment on mouse lung epithelial 12 (MLE-12) cell was applied to establish an in vitro model of LPS-induced ALI. H&E staining, lung wet-to-dry ratio, and total broncho-alveolar lavage fluid (BALF) concentrations were used to evaluate lung injury. Overall, the m6A level was determined with the m6A RNA Methylation Quantification Kit and dot blot assay. Expression of METTL3 and neprilysin were measured with immunohistochemistry, immunofluorescence, immunofluorescence-fluorescence in situ hybridization, and western blot. Apoptosis was detected with TUNEL, western blot, and flow cytometry. The interaction of METTL3 and neprilysin was determined with RIP-qPCR and MeRIP. Key findingsMETTL3 expression and apoptosis were increased in alveolar epithelial cells of mice treated with LPS, and METTL3-CKO or METTL3 inhibitor STM2457 could alleviate apoptosis and LPS-induced ALI. In MLE-12 cells, LPS-Induced METTL3 expression and apoptosis. Knockdown of METTL3 alleviated, while overexpression of METTL3 exacerbated LPS-induced apoptosis. LPS treatment reduced neprilysin expression, the intervention of neprilysin expression negatively regulated apoptosis without affecting METTL3 expression, and mitigated the promoting effect of METTL3 on LPS-induced apoptosis. Additionally, METTL3 could bind to the mRNA of neprilysin, and reduce its expression. SignificanceOur findings revealed that inhibition of METTL3 could exert anti-apoptosis and ALI-protective effects via restoring neprilysin expression.

METTL14-Mediated m6A Modification of TNFAIP3 Involved in Inflammation in Patients With Active Rheumatoid Arthritis.

The aim of the study was to investigate the role of N6 -methyladenosine (m6A) modification in the progression of rheumatoid arthritis (RA). Peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls were collected. The expression of m6A modification-related proteins and m6A levels were detected using polymerase chain reaction (PCR), western blot, and m6A enzyme-linked immunosorbent assay (ELISA). The roles of methyltransferase-like 14 (METTL14) in the regulation of inflammation in RA was explored using methylated RNA immunoprecipitation (MeRIP) sequencing and RNA immunoprecipitation assays. Collagen antibody-induced arthritis (CAIA) mice were used as an in vivo model to study the role of METTL14 in the inflammation progression of RA. We found that m6A writer METTL14 and m6A levels were decreased in PBMCs of patients with active RA and correlated negatively with the disease activity score using 28 joint counts (DAS28). Knockdown of METTL14 downregulated m6A and promoted the secretion of inflammatory cytokines interleukin 6 (IL-6) and IL-17 in PBMCs of patients with RA. Consistently, METTL14 knockdown promoted joint inflammation accompanied by upregulation of IL-6 and IL-17 in CAIA mice. MeRIP sequencing and functional studies confirmed that tumor necrosis factor α induced protein 3 (TNFAIP3), a key suppressor of the nuclear factor-κB inflammatory pathway, was involved in m6A-regulated PBMCs. Mechanistic investigations revealed that m6A affected TNFAIP3 expression by regulation of messenger RNA stability and translocation in TNFAIP3 protein coding sequence. Our study highlights the critical roles of m6A on regulation of inflammation in RA progression. Treatment strategies targeting m6A modification may represent a new option for management of RA.

METTL3 promotes drug resistance to oxaliplatin in gastric cancer cells through DNA repair pathway.

Gastric cancer (GC) poses a significant threat to human health and remains a prevalent form of cancer. Despite clinical treatments, the prognosis for Gastric cancer patients is still unsatisfactory, largely due to the development of multidrug resistance. Oxaliplatin (OXA), a second-generation platinum drug, is commonly recommended for adjuvant and palliative chemotherapy in Gastric cancer; however, the underlying mechanisms of acquired resistance to Oxaliplatin in Gastric cancer patients are not yet fully understood. In this study, we aimed to explore the potential mechanisms of Oxaliplatin resistance in Gastric cancer by employing bioinformatics analysis and conducting in vitro experiments. Specifically, we focused on investigating the role of methyltransferase-like 3 (METTL3). Our findings revealed that the knockdown of METTL3 significantly impeded the proliferation and migration of Gastric cancer cells. METTL3 knockdown induced apoptosis in OXA-resistant Gastric cancer cells and enhanced their sensitivity to Oxaliplatin. Furthermore, we found that DNA repair pathways were significantly activated in OXA-resistant Gastric cancer cells, and METTL3 knockdown significantly inhibited DNA repair pathways. Another important finding is that METTL3 knockdown and OXA-induced Gastric cancer cell death are additive, and the targeted METTL3 can assist Oxaliplatin treatment. Collectively, our findings suggest that METTL3 knockdown can augment the sensitivity of Gastric cancer cells to Oxaliplatin by impeding DNA repair processes. Consequently, targeting METTL3 holds great promise as a viable adjuvant strategy in the treatment of Gastric cancer patients.

METTL3 promotes proliferation and migration of colorectal cancer cells by increasing SNHG1 stability.

N6‑methyladenosine (m6A) serves an essential role in RNA modulation and is implicated in multiple malignancies, including colorectal cancer (CRC). Methyltransferase‑like 3 (METTL3) is an important writer in m6A modification, however its role in CRC in modifying small nucleolar RNA host gene 1 (SNHG1), an oncogenic long noncoding RNA, remains unclear. In the present study, METTL3 expression in CRC was assessed using online bioinformatics analysis, immunohistochemistry staining, western blotting, reverse transcription (RT)‑quantitative PCR (qPCR) and cell transfections. Cell proliferation, migration and invasion were determined using functional Cell Counting Kit‑8 (CCK‑8) and Transwell assays. SNHG1 expression in CRC was evaluated using online bioinformatics analysis and RT‑qPCR. Methylated RNA immunoprecipitation qPCR was performed to assess m6A modification changes of SNHG1 mRNA. The present study demonstrated that METTL3 is upregulated in CRC tissues and cell lines. Moreover, METTL3 expression was associated with several unfavourable clinical features in patients with CRC, including the stage of lymph node metastases and overall survival. Functional Transwell and CCK‑8 assays demonstrated that knockdown of METTL3 suppressed CRC cell proliferation and migration. Furthermore, METTL3 was positively correlated with SNHG1 in CRC tissue, as indicated by analysis of data from The Cancer Genome Atlas. Mechanistically, SNHG1 contains 18 m6A modification sites. Through cell transfectionsand actinomycin D assays, the present study found that METTL3‑mediated m6A modification at these sites enhances the stability of SNHG1 in CRC cells. Finally, it was demonstrated that SNHG1 knockdown partially diminished the facilitative effect of METTL3 on CRC cell migration and proliferation. The present study concluded that METTL3, a potential biomarker for assessing overall survival and metastasis in CRC, may serve as an oncogene, promote SNHG1 m6A modification, improve the stability of SNHG1 and enhance SNHG1‑mediated oncogenic function in CRC.

Methyltransferase-like 3 facilitates the stem cell properties of esophageal cancer by upregulating patched homolog 1 via N6-methyladenosine methylation.

Patched homolog 1 (PTCH1) has been proven to facilitate cell proliferation and self-renewal in esophageal cancer (EC). The present study intended to exploit the influence of PTCH1 on EC cells and the potential mechanisms. PTCH1 and methyltransferase-like 3 (METTL3) expression were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot in EC cell lines. Following the loss- and gain-of-function assays, cell proliferation was examined by cell counting kit (CCK)-8 and clone formation assays, invasion and migration by Transwell and scratch assays, and the sphere-forming ability of stem cells by cell sphere-forming assay. The expression of stemness genes NANOG homeobox protein (NANOG), octamer-binding transcription factor 4 (Oct4), and sex-determining region Y-box 2 (SOX2) was detected by Western blot. Methylated RNA immunoprecipitation (Me-RIP) assay was performed to test N6-methyladenosine (m6A) modification levels of PTCH1 mRNA, RIP and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) assays to assess the binding of METTL3 to PTCH1, and actinomycin D treatment to examine PTCH1 mRNA stability. A xenograft tumor model in nude mice was established for further in vivo verification. PTCH1 and METTL3 expression was high in EC cells. Knockdown of METTL3 reduced m6A level and stability of PTCH1 mRNA. Knockdown of PTCH1 or METTL3 declined invasion, proliferation, migration, and NANOG, Oct4, and SOX2 levels in EC cells, and reduced sphere-forming abilities of EC stem cells. Overexpression of PTCH1 abolished the suppressive effect of METTL3 knockdown on EC cells in vitro. METTL3 knockdown repressed tumor growth in nude mice, which was negated by further overexpressing PTCH1. METTL3 facilitated growth and stemness of EC cells via upregulation of PTCH1 expression by enhancing PTCH1 m6A modification.NEW & NOTEWORTHY PTCH1 has been proved to facilitate cell proliferation and self-renewal in esophageal cancer. We studied the upstream regulation mechanism of PTCH1 by METTL3 through m6A modification. Our results provide a new target and theoretical basis for the treatment of esophageal cancer.

Decreased expression of Chrna4 by METTL3-mediated m6A modification participates in BPA-induced spatial memory deficit

Bisphenol A (BPA), a widely used endocrine disruptor, has been implicated in cognitive impairment via epigenetic machinery. N6-methyl adenosine (m6A) has recently emerged as a new epigenetic factor that influences cognition, but the role of m6A in BPA induced cognitive deficits has not been explored yet. In this study, we found increased global m6A abundance accompanied with elevated expression of methyltransferase-like 3 (METTL3) in hippocampal neurons following BPA exposure. Inhibition of METTL3 activity by selective METTL3 inhibitor 2457 (STM) in cultured neurons abolished BPA induced m6A upregulation and abnormal synaptic transmission. Additionally, knockdown of METTL3 in hippocampus abrogated BPA induced learning and memory deficit in rats. Further study showed that m6A modification was enriched in mRNA of cholinergic receptor nicotinic alpha 4 subunit (Chrna4). Inhibition of METTL3 either by STM or shRNA restored BPA induced downregulation of Chrna4, suggesting that Chrna4 may be a potential target involved in BPA induced neurotoxicity that modified by m6A. Collectively, our findings demonstrated that METTL3 mediated m6A modification was involved in BPA induced cognitive deficit with Chrna4 as a potential target, which enriched our understanding of the role of epigenetics (RNA modifications) in BPA induced neurotoxicity and provided new insights into BPA or its substitutes induced damages in other organs.

METTL3-Modulated circUHRF2 Promotes Colorectal Cancer Stemness and Metastasis through Increasing DDX27 mRNA Stability by Recruiting IGF2BP1.

Increasing evidence has implicated that circular RNAs (circRNAs) exert important roles in colorectal cancer (CRC) occurrence and progression. However, the role of a novel circRNA, circUHRF2, remains unknown in CRC. Our work aimed at identifying the functional roles of circUHRF2 in CRC and illustrating the potential mechanisms. As assessed by quantitative real-time PCR (qRT-PCR), circUHRF2 and methyltransferase-like 3 (METTL3) were highly expressed in CRC specimens and cells. Sanger sequencing and RNase R assays were performed to verify the ring structure of circUHRF2. Notably, aberrantly increased expression of circUHRF2 was positively correlated with poor prognosis of CRC patients. Functional experiments indicated that CRC stemness, migration, and epithelial-mesenchymal transition (EMT) were suppressed by the knockdown of circUHRF2 or METTL3. Mechanistically, METTL3 enhanced circUHRF2 expression through N6-methyladenine (m6A) modification. Rescue experiments showed that overexpression of circUHRF2 reversed the repressive effect of METTL3 silencing on CRC progression. Moreover, circUHRF2 inhibited the loss of DEAD-box helicase 27 (DDX27) protein via promoting the interaction between insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and DDX27 mRNA. DDX27 knockdown repressed CRC malignant properties, which was counteracted by circUHRF2 overexpression. The in vivo assays in nude mice demonstrated that circUHRF2 or METTL3 silencing exerted a suppressive effect on CRC growth and liver metastasis via repressing DDX27 protein expression. Taken together, METTL3-mediated m6A modification upregulated circUHRF2 and subsequently inhibited loss of DDX27 protein via recruitment of IGF2BP1, which conferred CRC stemness and metastasis. These findings shed light on CRC pathogenesis and suggest circUHRF2 as a novel target for CRC treatment.