Methyltransferase-like 3 Research Articles

Decreased expression of the m6A RNA methyltransferase METTL3 is associated with residual ridge resorption

ObjectiveN6-methyladenosine (m6A) methylation and its regulators play crucial roles in the progression of osteoporosis (OP) by regulating the expression of osteoporosis-related genes. In this study, we have analyzed the expression of methyltransferase-like 3 (METTL3) and its target gene Runt-related transcription factor 2 (RUNX2) in patients with residual ridge resorption (RRR). Materials and methodsA total 50 number of participants were included in this comparative study (RRR – n25 and healthy control – n25). Total RNA was extracted from peripheral blood and converted into cDNA. METTL3 and RUNX2 expression levels were quantified using RT-qPCR with GAPDH as the reference gene. Bioinformatics tools were used to identify gene functions and pathways. ResultsReal-time polymerase chain reaction (qPCR) revealed that METTL3 and RUNX2 expression was downregulated in the RRR group compared to that in healthy controls (P < 0.05). In silico functional analysis provided information regarding the role of METTL3 in various biological processes. ConclusionOur findings suggest that METTL3 dysregulation contributes to RRR pathogenesis. Further large-scale samples and functional studies are required to identify their therapeutic potential.

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.

Targeting histone deacetylase suppresses tumor growth through eliciting METTL14-modified m6 A RNA methylation in ocular melanoma.

Diversified histone deacetylation inhibitors (HDACis) have demonstrated encouraging outcomes in multiple malignancies. N6-methyladenine (m6 A) is the most prevalent messenger RNA modification that plays an essential role in the regulation of tumorigenesis. Howbeit, an in-depth understanding of the crosstalk between histone acetylation and m6 A RNA modifications remains enigmatic. This study aimed to explore the role of histone acetylation and m6 A modifications in the regulation of tumorigenesis of ocular melanoma. Histone modification inhibitor screening was used to explore the effects of HDACis on ocular melanoma cells. Dot blot assay was used to detect the global m6 A RNA modification level. Multi-omics assays, including RNA-sequencing, cleavage under targets and tagmentation, single-cell sequencing, methylated RNA immunoprecipitation-sequencing (meRIP-seq), and m6 A individual nucleotide resolution cross-linking and immunoprecipitation-sequencing (miCLIP-seq), were performed to reveal the mechanisms of HDACis on methyltransferase-like 14 (METTL14) and FAT tumor suppressor homolog 4 (FAT4) in ocular melanoma. Quantitative real-time polymerase chain reaction (qPCR), western blotting, and immunofluorescent staining were applied to detect the expression of METTL14 and FAT4 in ocular melanoma cells and tissues. Cell models and orthotopic xenograft models were established to determine the roles of METTL14 and FAT4 in the growth of ocular melanoma. RNA-binding protein immunoprecipitation-qPCR, meRIP-seq, miCLIP-seq, and RNA stability assay were adopted to investigate the mechanism by which m6 A levels of FAT4 were affected. First, we found that ocular melanoma cells presented vulnerability towards HDACis. HDACis triggered the elevation of m6 A RNA modification in ocular melanoma. Further studies revealed that METTL14 served as a downstream candidate for HDACis. METTL14 was silenced by the hypo-histone acetylation status, whereas HDACi restored the normal histone acetylation level of METTL14, thereby inducing its expression. Subsequently, METTL14 served as a tumor suppressor by promoting the expression of FAT4, a tumor suppressor, in a m6 A-YTH N6-methyladenosine RNA-binding protein 1-dependent manner. Taken together, we found that HDACi restored the histone acetylation level of METTL14 and subsequently elicited METTL14-mediated m6 A modification in tumorigenesis. These results demonstrate that HDACis exert anti-cancer effects by orchestrating m6 A modification, which unveiling a "histone-RNA crosstalk" of the HDAC/METTL14/FAT4 epigenetic cascade in ocular melanoma.

Fibroblast-specific knockout of METTL1 attenuates myocardial infarction-induced cardiac fibrosis

Cardiac fibrosis, a common pathology in inherited and acquired heart diseases, necessitates the identification of diagnostic and therapeutic targets. Methyltransferase Like 1 (METTL1), an enzyme responsible for RNA modification by methylating guanosine to form m7G, is an emerging area of research in understanding cellular processes and disease pathogenesis. Dysregulation of m7G modification has been implicated in various diseases. However, the role of METTL1 in cardiac fibrosis remains unclear. This study aimed to investigate the role of METTL1 in myocardial infarction-induced heart failure and cardiac fibrosis. Our findings demonstrate that elevated METTL1-mediated RNA m7G methylation is observed in cardiac fibrosis tissues and TGF-β1-induced cardiac fibroblast proliferation and myofibroblast transformation. Furthermore, fibroblast-specific knockout of METTL1 attenuated myocardial infarction-induced heart failure and cardiac fibrosis. Additionally, METTL1 knockout decreased m7G methylated fibrotic genes and impaired their translation efficiency. These results suggest a novel pro-fibrosis role of METTL1-mediated RNA m7G methylation, highlighting its potential as a therapeutic target in cardiac fibrosis.

METTL3-mediated m6A modification of pri-miR-148a-3p affects prostate cancer progression by regulating TXNIP.

Prostate cancer (PCa) severely affects men's health worldwide. The mechanism of methyltransferase-like 3 (METTL3) in affecting PCa development by regulating miR-148a-3p expression via N6-methyladenosine (m6A) modification was investigated. METTL3, miR-148a-3p, and thioredoxin interacting protein (TXNIP) levels were determined using RT-qPCR and Western blotting. The m6A modification level of miR-148a-3p was observed by Me-RIP assay. Bioinformatics website predicted miR-148a-3p and TXNIP levels in PCa and their correlation, and the binding site between them was verified by dual-luciferase assay. The proliferation, migration, invasion, and apoptosis of PCa cells were examined by CCK-8 assay, Transwell assay, and flow cytometry. A transplanted tumor model was established in nude mice to observe the tumor growth ability, followed by determination of TXNIP levels in tumor tissues by immunohistochemistry. METTL3 interference restrained the proliferation, migration, and invasion and promoted apoptosis of PCa cells. METTL3 up-regulated miR-148a-3p by promoting the m6A modification of pri-miR-148a-3p in PCa cells. miR-148a-3p overexpression nullified the inhibitory actions of silencing METTL3 on PCa cell growth. miR-148a-3p facilitated PCa cell growth by silencing TXNIP. METTL3 interference inhibited tumor growth by down-regulating miR-148a-3p and up-regulating TXNIP. METTL3 promoted miR-148a-3p by mediating the m6A modification of pri-miR-148a-3p, thereby targeting TXNIP, interfering with METTL3 to inhibit the proliferation, migration and invasion of PCa cells, promote apoptosis, and inhibit tumor growth in nude mice.

N6-Methyladenosine Directly Regulates CD40L Expression in CD4+ T Lymphocytes.

T cell activation is a highly regulated process, modulated via the expression of various immune regulatory proteins including cytokines, surface receptors and co-stimulatory proteins. N6-methyladenosine (m6A) is an RNA modification that can directly regulate RNA expression levels and it is associated with various biological processes. However, the function of m6A in T cell activation remains incompletely understood. We identify m6A as a novel regulator of the expression of the CD40 ligand (CD40L) in human CD4+ lymphocytes. Manipulation of the m6A 'eraser' fat mass and obesity-associated protein (FTO) and m6A 'writer' protein methyltransferase-like 3 (METTL3) directly affects the expression of CD40L. The m6A 'reader' protein YT521-B homology domain family-2 (YTHDF2) is hypothesized to be able to recognize and bind m6A specific sequences on the CD40L mRNA and promotes its degradation. This study demonstrates that CD40L expression in human primary CD4+ T lymphocytes is regulated via m6A modifications, elucidating a new regulatory mechanism in CD4+ T cell activation that could possibly be leveraged in the future to modulate T cell responses in patients with immune-related diseases.

METTL14 mediates m6a modification on osteogenic proliferation and differentiation of bone marrow mesenchymal stem cells by regulating the processing of pri-miR-873.

N6-methyl-adenosine (m6a) is involved in the occurrence and development of various diseases such as autogenic immune disease and tumors. Methyltransferases regulate primary (pri)-microRNA (miRNA/miR) processing by mediating m6a modifications, consequently affecting pathological processes including immune-related diseases by regulating both innate and adaptive immune cells. However, the roles of m6a on the biological functions of bone marrow mesenchymal stem cells (BMSCs) remain to be elucidated. The relative expression levels of methyltransferase-like 14 (METTL14) and other methyltransferases, demethylases, and miR-873 in bone samples from patients with osteoporosis and from normal individuals were measured by reverse transcription-quantitative PCR. Cell Counting Kit-8 assay was used to examine the proliferation of BMSCs. Co-immunoprecipitation (Co-IP) was used to investigate the binding of METTL14 to DiGeorge syndrome critical region 8 (DGCR8). RNA immunoprecipitation (RIP) was used to examine the binding of METTL14 to pri-miR-873. METTL14 and m6a modifications were highly detected in patients with osteoporosis compared with the controls. Co-IP results indicated that silencing of METTL14 reduced METTL14 and m6a modification levels in BMSCs. Downregulation of METTL14 significantly promoted the proliferation of BMSCs. RIP results suggested that METTL14/m6a methylation modification promoted the processing of pri-miR-873 by binding to DGCR8 in BMSCs. Furthermore, overexpression of miR-873 inhibited the proliferation of BMSCs. The results also showed that miR-873 mimics significantly inhibited the proliferation in small interfering (si)-METTL14 transfected BMSCs; however, miR-873 inhibitors markedly promoted the proliferation of si-METTL14 transfected BMSCs. METTL14 and m6a modifications were upregulated in osteoporosis samples. METTL14 promoted the processing of pri-miR-873 into mature miR-873 by regulating m6a modification. Furthermore, overexpression of miR-873 significantly inhibited the proliferation of BMSCs. Therefore, the METTL14/m6a/miR-873 axis may be a potential target for the treatment of osteoporosis.

Methyltransferase like 3 inhibition limits intrahepatic cholangiocarcinoma metabolic reprogramming and potentiates the efficacy of chemotherapy.

N6-methyladenosine (m6A) RNA methylation and its associated methyltransferase like 3 (METTL3) are involved in the development and maintenance of various tumors. The present study aimed to evaluate the cross-talk of METTL3 with glucose metabolism and reveal a novel mechanism for intrahepatic cholangiocarcinoma (ICC) progression. Real-time quantitative PCR, western blotting, and immunohistochemistry analyses suggested that METTL3 was highly expressed in ICC, which was correlated with poor patient prognosis. Immunoprecipitation sequencing of m6A-RNA showed that METTL3 upregulated m6A modification of NFAT5, which recruited IGF2BP1 for NFAT5 mRNA stabilization. Elevated expression of NFAT5 increased the expression of the gluconeogenesis-related genes GLUT1 and PGK1, resulting in enhanced aerobic glycolysis, proliferation, and tumor metastasis of ICC. Moreover, higher METTL3 expression was observed in tumor tissues of ICC patients with activated ICC glucose metabolism. Importantly, STM2457, a highly potent METTL3 inhibitor, which inhibited METTL3 activity and acted synergistically with gemcitabine, suggests that reprogramming RNA epigenetic modifications may serve as a potential therapeutic strategy. Overall, our findings highlighted the role of METTL3-mediated m6A modification of NFAT5 in activating glycolytic reprogramming in ICC and proposed that the METTL3/NFAT5 axis was a clinical target for the management of ICC chemoresistance by targeting cancer glycolysis.

Modulation of miR-146b by N6-methyladenosine modification remodels tumor-associated macrophages and enhances anti-PD-1 therapy in colorectal cancer

PurposeMicroRNA-146b (miR-146b) alleviates experimental colitis in mice by mediating macrophage polarization and the release of inflammatory factors. Our goals were to evaluate the antitumor efficacy of miR-146b in colorectal cancer (CRC) and to investigate the underlying mechanisms.MethodsWe used murine models of CRC to evaluate whether miR-146b influenced the progression of tumors independent of tumor-associated macrophages (TAMs). RNA immunoprecipitation, N6-methyladenosine (m6A) RNA immunoprecipitation and in vitro pri-miRNA processing assays were conducted to examine whether m6A mediates the maturation of pri-miR-146b/miR-146b. In a series of in vitro and in vivo experiments, we further defined the molecular mechanisms of methyltransferase-like 3 (METTL3)/miR-146b-mediated antitumor immunity and its efficacy in combination with anti-PD-1 immunotherapy.ResultsWe found that miR-146b deletion supported tumor progression by increasing the number of alternatively activated (M2) TAMs. Mechanistically, the m6A-related “writer” protein METTL3 and “reader” protein HNRNPA2B1 controlled miR-146b maturation by regulating the m6A modification region of pri-miR-146b. Furthermore, miR-146b deletion promoted the polarization of M2-TAMs by enhancing phosphoinositide 3-kinase (PI3K)/AKT signaling, and this effect was mediated by the class IA PI3K catalytic subunit p110β, which reduced T cell infiltration, aggravated immunosuppression and ultimately promoted tumor progression. METTL3 knockdown or miR-146b deletion induced programmed death ligand 1 (PD-L1) production via the p110β/PI3K/AKT pathway in TAMs and consequently augmented the antitumor activity of anti-PD-1 immunotherapy.ConclusionsThe maturation of pri-miR-146b is m6A-dependent, and miR-146b deletion-mediated TAM differentiation promotes the development of CRC by activating the PI3K/AKT pathway, which induces upregulation of PD-L1 expression, inhibits T cell infiltration into the TME and enhances the antitumor activity of anti-PD-1 immunotherapy. The findings reveal that targeting miR-146b can serve as an adjuvant to anti-PD-1 immunotherapy.Graphical

M6A-mediated upregulation of miRNA-193a aggravates cardiomyocyte apoptosis and inflammatory response in sepsis-induced cardiomyopathy via the METTL3/ miRNA-193a/BCL2L2 pathway

The impact of N6-methyladenosine (m6A) modification on pri-miRNA in sepsis-induced cardiomyopathy (SICM), and its underlying regulatory mechanism, have not been fully elucidated. We successfully constructed a SICM mice model through cecal ligation and puncture (CLP). In vitro, a lipopolysaccharide (LPS)-induced HL-1 cells model was also established. The results showed that sepsis frequently resulted in excessive inflammatory response concomitant with impaired myocardial function in mice exposed to CLP, as indicated by decreases in ejection fraction (EF), fraction shortening (FS), and left ventricular end diastolic diameters (LVDd). miR-193a was enriched in CLP mice heart and in LPS-treated HL-1 cells, while overexpression of miR-193a significantly increased the expression levels of cytokines. Sepsis-induced enrichment of miR-193a significantly inhibited cardiomyocytes proliferation and enhanced apoptosis, while this was reversed by miR-193a knockdown. Furthermore, under our experimental conditions, enrichment of miR-193a in SICM could be considered excessively maturated on pri-miR-193a by enhanced m6A modification. This modification was catalyzed by sepsis-induced overexpression of methyltransferase-like 3 (METTL3). Moreover, mature miRNA-193a bound to a predictive sequence within 3′UTRs of a downstream target, BCL2L2, which was further validated by the observation that the BCL2L2-3′UTR mutant failed to decrease luciferase activity when co-transfected with miRNA-193a. The interaction between miRNA-193a and BCL2L2 resulted in BCL2L2 downregulation, subsequently activating the caspase-3 apoptotic pathway. In conclusion, sepsis-induced miR-193a enrichment via m6A modification plays an essential regulatory role in cardiomyocyte apoptosis and inflammatory response in SICM. The detrimental axis of METTL3/m6A/miR-193a/BCL2L2 is implicated in the development of SICM.

Epigenetic regulatory mechanism of ADAMTS12 expression in osteoarthritis

BackgroundOsteoarthritis (OA) is a degenerative joint disease with lacking effective prevention targets. A disintegrin and metalloproteinase with thrombospondin motifs 12 (ADAMTS12) is a member of the ADAMTS family and is upregulated in OA pathologic tissues with no fully understood molecular mechanisms.MethodsThe anterior cruciate ligament transection (ACL-T) method was used to establish rat OA models, and interleukin-1 beta (IL-1β) was administered to induce rat chondrocyte inflammation. Cartilage damage was analyzed via hematoxylin-eosin, Periodic Acid-Schiff, safranin O-fast green, Osteoarthritis Research Society International score, and micro-computed tomography assays. Chondrocyte apoptosis was detected by flow cytometry and TdT dUTP nick-end labeling. Signal transducer and activator of transcription 1 (STAT1), ADAMTS12, and methyltransferase-like 3 (METTL3) levels were detected by immunohistochemistry, quantitative polymerase chain reaction (qPCR), western blot, or immunofluorescence assay. The binding ability was confirmed by chromatin immunoprecipitation-qPCR, electromobility shift assay, dual-luciferase reporter, or RNA immunoprecipitation (RIP) assay. The methylation level of STAT1 was analyzed by MeRIP-qPCR assay. STAT1 stability was investigated by actinomycin D assay.ResultsThe STAT1 and ADAMTS12 expressions were significantly increased in the human and rat samples of cartilage injury, as well as in IL-1β-treated rat chondrocytes. STAT1 is bound to the promoter region of ADAMTS12 to activate its transcription. METTL3/ Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) mediated N6-methyladenosine modification of STAT1 promoted STAT1 mRNA stability, resulting in increased expression. ADAMTS12 expression was reduced and the IL-1β-induced inflammatory chondrocyte injury was attenuated by silencing METTL3. Additionally, knocking down METTL3 in ACL-T-produced OA rats reduced ADAMTS12 expression in their cartilage tissues, thereby alleviating cartilage damage.ConclusionMETTL3/IGF2BP2 axis increases STAT1 stability and expression to promote OA progression by up-regulating ADAMTS12 expression.

CircTTLL13 Promotes TMZ Resistance in Glioma via Modulating OLR1-Mediated Activation of the Wnt/β-Catenin Pathway.

Glioma, originating from neuroglial progenitor cells, is a type of intrinsic brain tumor with poor prognosis. temozolomide (TMZ) is the first-line chemotherapeutic agent for glioma. Exploring the mechanisms of circTTLL13 underlying TMZ resistance in glioma is of great significance to improve glioma treatment. Bioinformatics was adopted to identify target genes. The circular structure of circTTLL13 and its high expression in glioma cells were disclosed by quantitative real time-PCR (qRT-PCR) and PCR-agarose gel electrophoresis. Functional experiments proved that oxidized LDL receptor 1 (OLR1) promotes TMZ resistance of glioma cells. CircTTLL13 enhances TMZ resistance of glioma cells via modulating OLR1. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), RNA pulldown, mRNA stability, N6-methyladenosine (m6A) dot blot and RNA total m6A quantification assays were implemented, indicating that circTTLL13 stabilizes OLR1 mRNA via recruiting YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and promotes m6A methylation of OLR1 pre-mRNA through recruiting methyltransferase-like 3 (METTL3). TOP/FOP-flash reporter assay and western blot verified that circTTLL13 activates Wnt/β-catenin signaling pathway by regulating OLR1. CircTTLL13 promotes TMZ resistance in glioma through regulating OLR1-mediated Wnt/β-catenin pathway activation. This study offers an insight into the efficacy improvement of TMZ for glioma treatment.

Prognostic significance of N6-methyladenosine-modified related chemotransferase METTL3 in gastric carcinoma: Evidence from meta-analysis.

N6-methyladenosine (m6A) methylation is known as the research hotspot for tumor epimodification, and its associated methyltransferase-like3 (METTL3) is significantly differentially expressed in gastric carcinoma, but its clinical value has not been summarized. This meta-analysis aimed to evaluate the prognostic significance of METTL3 in gastric carcinoma. Databases, including PubMed, EMBASE (Ovid platform), Science Direct, Scopus, MEDLINE, Google Scholar, Web of Science, and Cochrane Library, were used to identify relevant eligible studies. The endpoints included overall survival, progression-free survival, recurrence-free survival, post-progression survival, and disease-free survival. Hazard ratios (HR) with 95% confidence intervals (CI) were used to correlate METTL3 expression with prognosis. Subgroup and sensitivity analyses were performed. Seven eligible studies involving 3034 gastric carcinoma patients were recruited for this meta-analysis. The analysis showed that high METTL3 expression was associated with significantly poorer overall survival (HR = 2.37, 95% CI 1.66-3.39, P < 0.01) and unfavorable disease-free survival (HR = 2.58, 95% CI 1.97-3.38, P < 0.01), as did unfavorable progression-free survival (HR = 1.48, 95% CI 1.19-1.84, P < 0.01)/recurrence-free survival (HR = 2.62, 95% CI 1.93-5.62, P < 0.01)/post-progression survival (HR = 1.53, 95% CI 1.22-1.91, P < 0.01). Subgroup analysis found that high METTL3 expression was associated with worse overall survival in patients with Chinese (HR = 2.21, 95% CI 1.48-3.29, P < 0.01), in studies with sample source from formalin-fixed, paraffin-embedded tissues (HR = 2.66, 95% CI 1.79-3.94, P < 0.01), and the reported directly from articles group (HR = 2.42, 95% CI 1.66-3.53, P < 0.01). The subgroup analysis that was performed based on sample size, detected method, and follow-up showed the same results. High expression of METTL3 predicts poor prognosis in gastric carcinoma, indicating promise for METTL3 as a prognostic biomarker.Systematic review registration: https://www.crd.york.ac.uk/prospero, ID = CRD42023408519.

Role of METTL3-mediated m6A modification in osteogenic differentiation of periodontal ligament stem cells extracted from adult periodontal ligaments ex-vivo.

Periodontal ligament stem cells (PDLSCs) are identified as candidate cells for the regeneration of periodontal and alveolar bone tissues. This research was to analyze the effect of methyltransferase-like 3 (METTL3)-mediated m6A modification on the osteogenic differentiation of PDLSCs extracted from adult periodontal ligaments (PDLs) ex-vivo. From June 2022 to October 2022, 27 patients undergoing orthodontic treatment in our hospital were selected as the research population, with 31 teeth extracted in total. PDLSCs were isolated from PDLs by tissue block culture, and the results were analyzed. Then PDLSCs were induced to differentiate into osteoblasts, and changes in METTL3 and m6A levels during differentiation were observed. Additionally, abnormal METTL3 expression vectors were constructed and transfected into PDLSCs to observe the influence of METTL3 on the biological behavior and osteogenic differentiation of PDLSCs. PDLSCs isolated from ex-vivo PDLs were predominantly spindle-shaped, with high CD73, CD90 and CD105 levels and low CD11b, CD34 and CD45 levels, showing the characteristics of stem cells. Spearman correlation coefficients identified a positive connection between Runx2, Sp7, Alp, Bglap, METTL3 and m6A levels and osteogenic differentiation incubation time (P<0.05). As METTL3 expression was increased, the proliferation capacity and osteogenic differentiation ability of PDLSCs were enhanced (P<0.05), and the content of m6A was increased (P<0.05). However, the activity and osteogenic differentiation ability of PDLSCs was decreased after silencing METTL3 (P<0.05). In conclusion, METTL3-mediated m6A modification promoted the osteogenic differentiation of PDLSCs extracted from adult PDLs ex vivo. This study offered a novel understanding of the mechanisms underlying osteogenic differentiation, and implied a possible method for accelerating bone formation.

METTL3-mediated m6A RNA modification promotes corneal neovascularization by upregulating the canonical Wnt pathway during HSV-1 infection

BackgroundCorneal neovascularization (CNV) is a symptom of herpes simplex keratitis (HSK), which can result in blindness. The corneal angiogenesis brought on by herpes simplex virus type 1 (HSV-1) is strongly affected by vascular endothelial growth factor A (VEGFA). The N6-methyladenosine (m6A) modification catalyzed by methyltransferase-like 3 (METTL3) is a crucial epigenetic regulatory process for angiogenic properties. However, the roles of METTL3 and m6A in HSK-induced CNV remain unknown. Here, we investigated these roles in vitro and in vivo. MethodsA PCR array in HSV-1-infected human umbilical vein endothelial cells (HUVECs) was used to screen for METTL3 among the epitranscriptomic genes. Tube formation and scratch assays were conducted to investigate cell migration capacity. The global mRNA m6A abundance was evaluated using a dot blot assay. Gene expression was assessed by RT-qPCR, western blotting, and fluorescence immunostaining. In addition, bioinformatic analysis was conducted to identify the downstream molecules of METTL3 in HUVECs. METTL3 knockdown and STM2457 treatment clarified the specific underlying molecular mechanisms affecting HSV-1-induced angiogenesis in vitro. An acute HSK mouse model was established to examine the effects of METTL3 knockdown or inhibition using STM2457 on pathological angiogenic development in vivo. ResultsMETTL3 was highly upregulated in HSV-1-infected HUVECs and led to increased m6A levels. METTL3 knockdown or inhibition by STM2457 further reduced m6A levels and VEGFA expression and impaired migration and tube formation capacity in HUVECs after HSV-1 infection. Mechanistically, METTL3 regulated LRP6 expression through post-transcriptional mRNA modification in an m6A-dependent manner, increasing its stability, upregulating VEGFA expression, and promoting angiogenesis in HSV-1-infected HUVECs. Furthermore, METTL3 knockdown or inhibition by STM2457 reduced CNV in vivo. ConclusionOur findings revealed that METTL3 promotes pathological angiogenesis through canonical Wnt and VEGF signaling in vitro and in vivo, providing potential pharmacological targets for preventing the progression of CNV in HSK.

A cancer-associated METTL14 mutation induces aberrant m6A modification, affecting tumor growth

The methyltransferase-like 3 (METTL3)-/METTL14-containing complex predominantly catalyzes N6-methyladenosine (m6A) modification, which affects mRNA stability. Although the METTL14 R298P mutation is found in multiple cancer types, its biological effects are not completely understood. Here, we show that the heterozygous R298P mutation promotes cancer cell proliferation, whereas the homozygous mutation reduces proliferation. Methylated RNA immunoprecipitation sequencing analysis indicates that the R298P mutation reduces m6A modification at canonical motifs. Furthermore, this mutation induces m6A modification at aberrant motifs, which is evident only in cell lines harboring the homozygous mutation. The aberrant recognition of m6A modification sites alters the methylation efficiency at surrounding canonical motifs. One example is c-MET mRNA, which is highly methylated at canonical motifs close to the aberrantly methylated sites. Consequently, c-MET mRNA is severely destabilized, reducing c-Myc expression and suppressing cell proliferation. These data suggest that the METTL14 R298P mutation affects target recognition for m6A modification, perturbing gene expression patterns and cell growth.

METTL3 regulates N6-methyladenosine modification of ANGPTL3 mRNA and potentiates malignant progression of stomach adenocarcinoma

BackgroundN6-methyladenosine (m6A) is associated with mammalian mRNA biogenesis, decay, translation and metabolism, and also contributes greatly to gastrointestinal tumor formation and development. Therefore, the specific mechanisms and signaling pathways mediated by methyltransferase-like 3 (METTL3), which catalyzes the formation of m6A chemical labeling in stomach adenocarcinoma (STAD), are still worth exploring.MethodsQuantitative real-time PCR (qRT-PCR) was constructed to detect the expression of METTL3 in gastric cancer cell lines and patient tissues. The biological function of METTL3 was investigated in vitro/in vivo by Cell Counting Kit-8, colony formation assay, Transwell assay and nude mouse tumorigenesis assay. Based on the LinkedOmics database, the genes co-expressed with METTL3 in the TCGA STAD cohort were analyzed to clarify the downstream targets of METTL3. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) and RNA stability analysis were employed to explore the mechanism of METTL3 in gastric cancer progression.ResultsWe analyzed TCGA data and found that METTL3 was frequently elevated in STAD, and demonstrated that METTL3 was present at high levels in clinical STAD tissues and cells. High METTL3 expression was more likely to have advanced TNM tumors and distant metastasis. On the other hand, METTL3 silencing effectively impeded the higher oncogenic capacity of AGS and HGC27 cells in vivo and in vitro, as reflected by slowed cell growth and diminished migration and invasion capacities. Continued mining of the TCGA dataset identified the co-expression of angiopoietin-like 3 (ANGPTL3) and METTL3 in STAD. Lower level of ANGPTL3 was related to increased level of METTL3 in STAD samples and shorter survival times in STAD patients. ANGPTL3 enrichment limited the growth and metastasis of STAD cells. Besides, ANGPTL3 mRNA levels could be decreased by METTL3-dominated m6A modifications, a result derived from a combination of MeRIP-qPCR and RNA half-life experiments. Importantly, the inhibitory effect of METTL3 silencing on cancer could be reversed to some extent by ANGPTL3 inhibition.ConclusionsOverall, our findings suggested that METTL3 functioned an oncogenic role in STAD by reducing ANGPTL3 expression in an m6A-dependent manner. The discovery of the METTL3-ANGPTL3 axis and its effect on STAD tumor growth will contribute to further studies on the mechanisms of gastric adenocarcinoma development.

Structure-Based Design of Inhibitors of the m6A-RNA Writer Enzyme METTL3.

Methyltransferase-like 3 (METTL3) and METTL14 form a heterodimeric complex that catalyzes the most abundant internal mRNA modification, N6-methyladenosine (m6A). METTL3 is the catalytic subunit that binds the co-substrate S-adenosyl methionine (SAM), while METTL14 is involved in mRNA binding. The m6A modification provides post-transcriptional level control over gene expression as it affects almost all stages of the mRNA life cycle, including splicing, nuclear export, translation, and decay. There is increasing evidence for an oncogenic role of METTL3 in acute myeloid leukemia. Here, we use structural and dynamic details of the catalytic subunit METTL3 for developing small-molecule inhibitors that compete with SAM. Starting from a hit identified by high-throughput docking, protein crystallography and molecular dynamics simulations were employed to guide the optimization of inhibitory activity. The potency was successfully improved by 8000-fold as measured by a homogeneous time-resolved fluorescence assay. The optimized compound is selective against the off-targets RNA methyltransferases METTL1 and METTL16.

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.

Increased METTL3 expression and m6A RNA methylation may contribute to the development of dry eye in primary Sjögren’s syndrome

BackgroundPrimary Sjögren’s syndrome (pSS) is a chronic autoimmune disorder defined by xerostomia and keratoconjunctivitis sicca, and its etiology remains unknown. N6-methyladenosine (m6A) is the predominant posttranscriptional modification in eukaryotic mRNAs and is dynamically regulated by m6A regulators. Dysregulation of m6A modification is closely associated with several autoimmune disorders, but the role of m6A modification in pSS remains unknown. This study investigated the potential role of m6A and m6A-related regulators in pSS patients with dry eye.MethodsThis cross-sectional study included forty-eight pSS patients with dry eye and forty healthy controls (HCs). Peripheral blood mononuclear cells (PBMCs) were isolated, and the level of m6A in total RNA was measured. The expression of m6A regulators was determined utilizing real-time PCR and western blotting. The serological indicators detected included autoantibodies, immunoglobulins (Igs), complement factors (Cs), and inflammatory indicators. Dry eye symptoms and signs were measured, including the ocular surface disease index, Schirmer’s test (ST), corneal fluorescein staining score (CFS), and tear break-up time. Spearman’s correlation coefficient was employed to assess the associations of m6A and m6A-related regulator expression with clinical characteristics.ResultsThe expression level of m6A was markedly increased in the PBMCs of pSS patients with dry eye compared to HCs (P value<0.001). The relative mRNA and protein expression levels of the m6A regulators methyltransferase-like 3 (METTL3) and YT521-B homology domains 1 were markedly elevated in pSS patients with dry eye (both P value<0.01). The m6A RNA level was found to be positively related to METTL3 expression in pSS patients (r = 0.793, P value<0.001). Both the m6A RNA level and METTL3 mRNA expression correlated with the anti-SSB antibody, IgG, ST, and CFS (all P values < 0.05). The m6A RNA level was associated with C4 (r = -0.432, P value = 0.002), while METTL3 mRNA expression was associated with C3 (r = -0.313, P value = 0.030).ConclusionsOur work revealed that the upregulation of m6A and METTL3 was associated with the performance of serological indicators and dry eye signs in pSS patients with dry eye. METTL3 may contribute to the pathogenesis of dry eye related to pSS.