METTL3 regulates cartilage development and homeostasis by affecting Lats1 mRNA stability in an m6A-YTHDF2-dependent manner.

Cartilage maintains the structure and function of joints, with disturbances leading to potential osteoarthritis. N6-methyladenosine (m6A), the most widespread post-transcriptional modification in eukaryotes, plays a crucial role in regulating biological processes. While current research has indicated that m6A affects the progression of osteoarthritis, its function in the development and homeostasis of articular cartilage remains unclear. Here we report that Mettl3 deficiency in chondrocytes leads to mandibular condylar cartilage morphological alterations, early temporomandibular joint osteoarthritis, and diminished adaptive response to abnormal mechanical stimuli. Mechanistically, METTL3 modulates Lats1 mRNA methylation and facilitates its degradation in an m6A-YTHDF2-dependent manner, which subsequently influences the degradation and nuclear translocation of YAP1. Intervention with the Hippo pathway inhibitor XMU-MP-1 alleviates condylar abnormality caused by Mettl3 knockout. Our findings demonstrate the role of METTL3 in cartilage development and homeostasis, offering insights into potential treatment strategies for osteoarthritis.

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

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

Anticancer effects of Erzhimaoling decoction in high-grade serous ovarian cancer in vitro and in vivo.

BACKGROUND: High-grade serous ovarian cancer (HGSOC) is a common gynecologic malignancy with a poor prognosis. The traditional Chinese medicine formula Erzhimaoling decoction (EZMLD) has anticancer potential. This study aims to elucidate the anticancer effects of EZMLD on HGSOC in vitro and in vivo. MATERIALS AND METHODS: EZMLD-containing serum was prepared from Sprague-Dawley rats for treating SKOV3 ovarian cancer cells at varying concentrations for 24 h and 48 h to determine the IC50. Concentrations of 0%, 5%, and 10% for 24 h were chosen for subsequent in vitro experiments. The roles of METTL3 and METTL14 in SKOV3 cells were explored by overexpressing these genes and combining EZMLD with METTL3/14 knockdown. Investigations focused on cell viability and apoptosis, apoptosis-related protein expression, and KRT8 mRNA m6A modification. For in vivo studies, 36 BALB/c nude mice were divided into six groups involving EZMLD (6.75, 13.5, and 27 g/kg) and METTL3 or METTL14 knockdowns, with daily EZMLD gavage for two weeks. RESULTS: In vitro, EZMLD-containing serum had IC50 values of 8.29% at 24 h and 5.95% at 48 h in SKOV3 cells. EZMLD-containing serum decreased SKOV3 cell viability and increased apoptosis. EZMLD upregulated METTL3/14 and FAS-mediated apoptosis proteins, while downregulating Keratin 8 (KRT8). EZMLD increased KRT8 mRNA m6A methylation. METTL3/14 overexpression reduced SKOV3 cell viability and increased apoptosis, while METTL3/14 knockdown mitigated EZMLD's effects. In vivo, EZMLD suppressed SKOV3 xenografts growth, causing significant apoptosis and modulating protein expression. CONCLUSIONS: EZMLD has therapeutic potential for ovarian cancer and may be considered for other cancer types. Future research may explore its broader effects beyond cell apoptosis.

METTL3 Inhibition Suppresses Cell Growth and Survival in Colorectal Cancer via ASNS Downregulation.

Background: Colorectal cancer (CRC) presents a significant global health burden, with high rates of incidence and mortality, and an urgent need to improve prognosis. STM2457, a novel small molecule inhibitor specific for N6-methyladenosine (m6A) catalytic enzyme Methyltransferase-like 3 (METTL3) has implicated significant treatment potentials in a few of types of cancer. However, its impact and underlying mechanism are still unclear in CRC cells. Methods: We used CCK-8 and colony formation assay to observe cell growth, flow cytometry and TUNEL approaches to detect cell apoptosis under the treatment of STM2457 on CRC cells in vitro or in vivo. RNA-sequencing, qRT-PCR and western blotting were performed to explore downstream effectors of STM2457. Messenger RNA stability was evaluated by qRT-PCR after treatment with actinomycin D. The methylated RNA immunoprecipitation (MeRIP) qPCR, dual-luciferase reporter analyses and m6A dot blotting were carried out to measure the m6A modification. Associated gene expression pattern and clinical relevance in CRC clinical tissue samples were analyzed using online database. Results: STM2457 exhibited a strong influence on cell growth suppression and apoptosis of CRC cells in vitro and subcutaneous xenograft growth in vivo. Asparagine synthetase (ASNS) was markedly downregulated upon STM2457 treatment or METTL3 knockdown and exogenous overexpression of ASNS could rescue the biological defects induced by STM2457. Mechanistically, the downregulation of ASNS by STM2457 may be due to the decrease of m6A modification level in ASNS mRNA mediated by METTL3. Conclusions: Our findings suggest that STM2457 may serve as a potential therapeutic agent and ASNS may be a new promising therapeutic target for CRC.

N6-methyladenosine-modified circ_0006168 promotes epithelial mesenchymal transition via miR-384/STAT3/Snail axis in esophageal squamous cell carcinoma.

Circular RNAs (circRNAs) are involved in the pathogenesis of esophageal squamous cell carcinoma (ESCC). This study aimed to explore the mechanisms of aberrant expression and functions of circ_0006168 in ESCC. In this study, real-time qPCR and fluorescence in situ hybridization (FISH) are adopted to estimate the expression and localization of circ_0006168 in cancer tissues and cells. Methylated RNA immunoprecipitation (MeRIP) was performed to detect the N6-methyladenosine (m6A) modification of circ_0006168. Gain- and loss-of-functions of circ_0006168 were performed to identify its role in ESCC progression. RNA-binding protein immunoprecipitation (RIP) was used to detect the interaction of circ_0006168 with IGF2BP2. Luciferase reporter assay and RIP are used to confirm the circ_0006168/miR-384/STAT3 ceRNA network. Our results showed that the expression of circ_0006168 was upregulated in ESCC tissues and cells. METTL3-mediated m6A modification increased the expression of circ_0006168 via IGF2BP2-dependent way in TE-1 cells. Circ_0006168 promoted cell proliferation, migration, invasion, cell cycle progression and inhibited cell apoptosis, while knockdown of circ_0006168 had the reverse effects. Mechanistically, circ_0006168 acted its functions via miR-384/STAT3/Snail axis in TE-1 cells. In conclusion, circ_0006168 is upregulated in ESCC and m6A methylation increased its expression via IGF2BP2. CircRNA_0006168 promotes cell migration, invasion by regulating EMT via miR-384/STAT3/Snail axis in ESCC.

METTL3 mediated ferroptosis in chondrocytes and promoted pain in KOA via HMGB1 m6A modification.

Methyltransferase-like 3 (METTL3) plays a role in the development of knee osteoarthritis (KOA). However, the mechanism underlying the role of METTL3 in KOA is unclear. This work investigated the effects of MELLT3 on ferroptosis and pain relief in in vitro and in vivo KOA models. Chondrocytes were treated with 10 ng/mL interleukin-1ß (IL-1ß) or 5 µM Erastin (ferroptosis inducer). IL-1ß or Erastin treatment inhibited cell viability and glutathione levels; increased Fe2+, lipid reactive oxygen species and malondialdehyde production; and decreased glutathione peroxidase 4, ferritin light chain and solute carrier family 7 member 11 levels. The overexpression of METTL3 facilitated the N6-methyladenosine methylation of high mobility group box 1 (HMGB1). HMGB1 overexpression reversed the effect of sh-METTL3 on IL-1ß-treated chondrocytes. A KOA rat model was established by the injection of monosodium iodoacetate into the joints and successful model establishment was confirmed by haematoxylin and eosin staining and Safranin O/Fast Green staining. METTL3 depletion alleviated cartilage damage, the inflammatory response, ferroptosis and knee pain in KOA model rats, and these effects were reversed by the addition of HMGB1. In conclusion, METTL3 depletion inhibited ferroptosis and the inflammatory response, and ameliorated cartilage damage and knee pain during KOA progression by regulating HMGB1.

LncRNA LINC00969 modified by METTL3 attenuates papillary thyroid cancer progression in an m6A-dependent manner.

BACKGROUND: The long non-coding RNA (lncRNA) LINC00969 is involved in human disease progression, and n6-methyladenosine (m6A) modification of lncRNAs in cancer has been proven to be a key regulatory mechanism. However, our understanding of its effects and mechanisms of action in papillary thyroid carcinoma (PTC) remains limited. OBJECTIVES: This study aimed to elucidate the role of methyltransferase-like 3 (METTL3)-induced m6A modification of LINC00969 in PTC tumorigenesis. MATERIAL AND METHODS: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to analyze LINC00969 and METTL3 mRNA levels in PTC. The regulation of LINC00969 by METTL3 was confirmed using cell function experiments, molecular biology assays and bioinformatics analysis. LINC00969 stabilization analysis was performed to verify the regulatory roles of METTL3 and LINC00969. RESULTS: LINC00969 expression was downregulated in PTC tissues. Increased LINC00969 expression inhibited the invasion, growth and migration of PTC cells. METTL3 downregulation in PTC mediated the m6A modification of LINC00969, increasing its stability. Furthermore, METTL3 levels were downregulated in PTC, and its silencing partially reversed the inhibitory effect of LINC00969 overexpression on PTC cell malignancy. CONCLUSIONS: LINC00969 overexpression inhibits PTC cell malignancy via METTL3-mediated m6A modification. These findings suggest that METTL3-m6A-LINC00969 is a promising therapeutic target for PTC.

Peptide degrader-based targeting of METTL3/14 improves immunotherapy response in cutaneous melanoma.

METTL3 has emerged as a promising therapeutic target in cancer treatment, although its oncogenic functions in melanoma development and potential for therapeutic targeting drug have not been fully explored. In this study, we define the oncogenic role of METTL3 in melanoma development and progression. Building on this insight, we examine our recently designed peptide inhibitor RM3, which targets the binding interface of METTL3/14 complex for disruption and subsequent ubiquitin-mediated proteasomal degradation via the E3 ligase STUB1. RM3 treatment reduces proliferation, migration, and invasion, and induces apoptosis in melanoma cells in vitro and in vivo. Subsequent transcriptomic analysis identified changes in immuno-related genes following RM3-mediated suppression of METTL3/14 N6-methyladenosine (m6A) methyltransferase activity, suggesting a potential for interaction with immunotherapy. A combination treatment of RM3 with anti-PD-1 antibody results in significantly higher beneficial tumor response in vivo, with a good safety profile. Collectively, these findings not only delineate the oncogenic role of METTL3 in melanoma but also showcase RM3, acting as a peptide degrader, as a novel and promising strategy for melanoma treatment.

H3K36 methyltransferase SMYD2 affects cell proliferation and migration in Hirschsprung's disease by regulating METTL3.

The pathogenesis of Hirschsprung's disease (HSCR) is complex. Recently, it has been found that histone modifications can alter genetic susceptibility and play important roles in the proliferation, differentiation and migration of neural crest cells. H3K36 methylation plays a significant role in gene transcriptional activation and expression, but its pathogenic mechanism in HSCR has not yet been studied. This study aimed to elucidate its role and molecular mechanism in HSCR. Western blot analysis, immunohistochemistry (IHC) and reverse transcription-quantitative PCR (RT‒qPCR) were used to investigate H3K36 methylation and methyltransferase levels in dilated and stenotic colon tissue sections from children with. We confirm that SMYD2 is the primary cause of differential H3K36 methylation and influences cell proliferation and migration in HSCR. Subsequently, quantitative detection of m6A RNA methylation revealed that SMYD2 can alter m6A methylation levels. Western blot analysis, RT-qPCR, co-immunoprecipitation (co-IP), and immunofluorescence colocalization were utilized to confirm that SMYD2 can regulate METTL3 expression and affect m6A methylation, affecting cell proliferation and migration. These results confirm that the H3K36 methyltransferase SMYD2 can affect cell proliferation and migration in Hirschsprung's disease by regulating METTL3. Our study suggested that H3K36 methylation plays an important role in HSCR, confirming that the methyltransferase SMYD2 can affect m6A methylation levels and intestinal nervous system development by regulating METTL3 expression.

ATF1 promotes ferroptosis resistance in lung cancer through enhancing mRNA stability of PROM2.

BACKGROUND: Ferroptotic proteins are promising therapeutic targets for lung cancer. The PROM2 is upregulated in lung cancer and known to suppress ferroptosis. This study examined the molecular mechanisms for PROM2-induced ferroptosis resistance in lung cancer. METHODS: Ferroptosis in lung cancer was assessed by iron kit, and transmission electron microscopy was applied to observe the changes in mitochondrial morphology. BODIPY™ was applied to test the lipid ROS, and MeRIP was performed to test the m6A modification of PROM2. RIP assay was employed for confirming the binding between METTL3 and PROM2. In addition, dual luciferase assay was employed for exploring the transcriptional regulation of ATF1 to METTL3, and the binding relation between ATF1 and METTL3 promoter region was explored by ChIP assay. RESULTS: Expression levels of PROM2 were significantly higher in lung cancer cell lines than a noncancerous control line, and PROM2 knockdown significantly reduced both cancer cell viability and proliferation rate. In addition, PROM2 knockdown reduced xenograft tumor growth and exacerbated erastin-induced ferroptosis. Compared to PROM2 mRNA from control cells, transcripts in lung cancer cells exhibited enhanced m6A levels, and showed greater binding with METTL3. Further, ATF1 upregulated METTL3 transcription, thereby stabilizing PROM2 mRNA and increasing ferroptosis resistance. CONCLUSION: ATF1 could promote ferroptosis resistance in lung cancer through enhancing mRNA stability of PROM2. Thus, our work might shed novel insights on discovering therapeutic strategy for lung cancer.

LINC01410 accelerates the invasion of trophoblast cells by modulating METTL3/Fas.

BACKGROUND: Insufficient trophoblast invasion, culminating in suboptimal uterine spiral artery remodeling, is pinpointed as a pivotal contributor to preeclampsia (PE) development. LINC01410 has been documented to be increased in various neoplasms, and is significantly associated with the invasive capabilities of tumor cells. Nonetheless, its function and the mechanisms in the pathogenesis of PE require further investigation. METHODS AND RESULTS: LINC01410 and methyltransferase-like 3 (METTL3) were ectopically expressed in HTR-8/Svneo cells via lentiviral transduction. Subsequently, the cells' invasive capabilities and apoptosis rates were evaluated employing Transwell assays and flow cytometry, respectively. The interplay between LINC01410 and METTL3, alongside the m6A methylation of FAS, was probed through RNA immunoprecipitation (RIP). Additionally, the association between FAS and METTL3 was elucidated via Coimmunoprecipitation (Co-IP) assays. The protein level of NF-κB, BAX, and BCL-2 in LINC01410-overexpressing cells was detected by Western blot. Our findings revealed that LINC01410 elevation increased the invasive ability of HTR-8/Svneo cells, directly impacting METTL3 then leading to its reduced expression. Conversely, heightened METTL3 expression mitigated invasiveness while enhancing apoptosis in these cells. Moreover, METTL3's interaction with FAS led to increased FAS expression, subject to m6A methylation. A surge in LINC01410 markedly decreased both mRNA and protein levels of FAS. Furthermore, LINC01410 overexpression significantly reduced NF-κB and BAX protein levels while augmenting BCL-2. CONCLUSIONS: Upregulation of LINC01410 expression promotes trophoblast cell invasion by inhibiting FAS levels through modified m6A alteration and suppressing the NF-κB pathway. These findings underscore the pivotal role of LINC01410 in regulating trophoblast cell invasion and propose it as a promising therapeutic strategy for preventing or alleviating PE. This offers valuable insights for the clinical treatment of PE, for which definitive targeted therapy methods are currently lacking.

PCAF-mediated acetylation of METTL3 impairs mRNA translation efficiency in response to oxidative stress.

METTL3 methylates RNA and regulates the fate of mRNA through its methyltransferase activity. METTL3 enhances RNA translation independently of its catalytic activity. However, the underlying mechanism is still elusive. Here, we report that METTL3 is both interacted with and acetylated at lysine 177 by the acetyltransferase PCAF and deacetylated by SIRT3. Neither the methyltransferase activity nor the stability of METTL3 is affected by its acetylation at K177. Importantly, acetylation of METTL3 blocks its interaction with EIF3H, a subunit of the translation initiation factor, thereby reducing mRNA translation efficiency. Interestingly, acetylation of METTL3 responds to oxidative stress. Mechanistically, oxidative stress enhances the interaction of PCAF with METTL3, increases METTL3 acetylation, and suppresses the interaction of METTL3 with EIF3H, thereby decreasing the translation efficiency of ribosomes and inhibiting cell proliferation. Altogether, we suggest a mechanism by which oxidative stress regulates RNA translation efficiency by the modulation of METTL3 acetylation mediated by PCAF.

METTL3/IGF2BP1 influences the development of non-small-cell lung cancer by mediating m6A methylation modification of TRPV1.

BACKGROUND: Methyltransferase 3 (METTL3) accelerates N6-methyladenosine (m6A) modifications and affects cancer progression, including non-small-cell lung cancer (NSCLC). In this study, we aimed to explore the regulatory mechanisms of METTL3 underling NSCLC. METHODS: Immunohistochemical assay, quantitative real-time polymerase chain reaction (qRT-PCR) assay, and western blot assay were conducted for gene expression. MTT assay and colony formation assay were performed to explore cell proliferation capacity. Cell apoptosis and THP-1 cell polarization were estimated by flow cytometry analysis. Cell migration and invasion capacities were evaluated by transwell assay. Methylated RNA immunoprecipitation assay, dual-luciferase reporter assay, actinomycin D treatment and RIP assay were performed to analyze the relationships of METTL3, insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), and transient receptor potential cation channel subfamily V member 1 (TRPV1). The functions of METTL3 and TRPV1 in vivo were investigated through establishing the murine xenograft model. RESULTS: TRPV1 expression was upregulated in NSCLC and related poor prognosis. TRPV1 silencing inhibited NSCLC cell growth and metastasis, induced NSCLC cell apoptosis, and repressed M2 macrophage polarization. The results showed that METTL3 and IGF2BP1 could regulate TRPV1 expression through m6A methylation modification. Moreover, METTL3 deficiency inhibited NSCLC cell growth, metastasis, and M2 macrophage polarization and facilitated NSCLC cell apoptosis, while TRPV1 overexpression restored the impacts. In addition, METTL3 knockdown restrained tumor growth in vivo via regulating TRPV1 expression. CONCLUSION: METTL3 bound to IGF2BP1 and enhanced IGF2BP1's m6A recognition of TRPV1 mRNA, thereby promoting NSCLC cell growth and metastasis, and inhibiting M2 macrophage polarization.

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

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

METTL3 promotes immature dental pulp stem cells-induced angiogenesis by regulating ETS1 mRNA stability in an m6A-HuR-dependent manner.

Angiogenesis serves as the determinate element of pulp regeneration. Dental pulp stem cell (DPSC) implantation can promote the regeneration of dental pulp tissue. Herein, the role of m6A methyltransferase methyltransferase-like 3 (METTL3) in regulating DPSCs-induced angiogenesis during pulp regeneration therapy was investigated. Cell DPSC viability, HUVEC migration, and angiogenesis ability were analyzed by CCK-8 assay, wound healing, Transwell assay, and tube formation assay. The global and EST1 mRNA m6A levels were detected by m6A dot blot and Me-RIP. The interactions between E26 transformation-specific proto-oncogene 1(ETS1), human antigen R(HuR), and METTL3 were analyzed by RIP assay. The relationship between METTL3 and the m6A site of ETS1 was performed by dual-luciferase reporter assay. ETS1 mRNA stability was examined with actinomycin D. Herein, our results revealed that human immature DPSCs (hIDPSCs) showed stronger ability to induce angiogenesis than human mature DPSCs (hMDPSCs), which might be related to ETS1 upregulation. ETS1 knockdown inhibited DPSCs-induced angiogenesis. Our mechanistic experiments demonstrated that METTL3 increased ETS1 mRNA stability and expression level on DPSCs in an m6A-HuR-dependent manner. ETS1 upregulation abolished sh-METTL3's inhibition on DPSCs-induced angiogenesis. METTL3 upregulation promoted DPSCs-induced angiogenesis by enhancing ETS1 mRNA stability in an m6A-HuR-dependent manner. This study reveals a new mechanism by which m6A methylation regulates angiogenesis in DPSCs, providing new insights for stem cell-based tissue engineering.

The methyltransferase METTL3 regulates endothelial cell proliferation and inflammation via m6A RNA methylation-mediated TRAF1 expression.

The imbalance of vascular endothelial cell homeostasis is the key mechanism for the progression of many vascular diseases. RNA modification, particularly N6-Methyladenosine (m6A), plays important function in numerous biological processes. Nevertheless, the regulatory function of m6A RNA methylation in endothelial dysfunction remains insufficiently characterized. In this study, we established that the m6A methyltransferase METTL3 is critical for regulating endothelial function. Functionally, depletion of METTL3 results in decreased endothelial cells proliferation, survival and inflammatory response. Conversely, overexpression of METTL3 elicited the opposite effects. Mechanistically, MeRIP-seq identified that METTL3 catalyzed m6A modification of TRAF1 mRNA and enhanced TRAF1 translation, thereby up-regulation of TRAF1 protein. Over-expression of TRAF1 successfully rescued the inhibition of proliferation and adhesion of endothelial cells due to METTL3 knockdown. Additionally, m6A methylation-mediated TRAF1 expression can be reversed by the demethylase ALKBH5. Knockdown of ALKBH5 upregulated the level of m6A and protein level of TRAF1, and also increased endothelial cells adhesion and inflammatory response. Collectively, our findings suggest that METTL3 regulates vascular endothelium homeostasis through TRAF1 m6A modification, suggesting that targeting the METTL3-m6A-TRAF1 axis may hold therapeutic potential for patients with vascular diseases.

METTL3 shapes m6A epitranscriptomic landscape for successful human placentation.

Methyltransferase-like 3 (METTL3), the catalytic enzyme of methyltransferase complex for m6A methylation of RNA, is essential for mammalian development. However, the importance of METTL3 in human placentation remains largely unexplored. Here, we show that a fine balance of METTL3 function in trophoblast cells is essential for successful human placentation. Both loss-of and gain-in METTL3 functions are associated with adverse human pregnancies. A subset of recurrent pregnancy losses and preterm pregnancies are often associated with loss of METTL3 expression in trophoblast progenitors. In contrast, METTL3 is induced in pregnancies associated with fetal growth restriction (FGR). Our loss of function analyses showed that METTL3 is essential for the maintenance of human TSC self-renewal and their differentiation to extravillous trophoblast cells (EVTs). In contrast, loss of METTL3 in human TSCs promotes syncytiotrophoblast (STB) development. Global analyses of RNA m6A modification and METTL3-RNA interaction in human TSCs showed that METTL3 regulates m6A modifications on the mRNA molecules of critical trophoblast regulators, including GATA2, GATA3, TEAD1, TEAD4, WWTR1, YAP1, TFAP2C and ASCL2, and loss of METTL3 leads to depletion of mRNA molecules of these critical regulators. Importantly, conditional deletion of Mettl3 in trophoblast progenitors of an early post-implantation mouse embryo also leads to arrested self-renewal. Hence, our findings indicate that METLL3 is a conserved epitranscriptomic governor in trophoblast progenitors and ensures successful placentation by regulating their self-renewal and dictating their differentiation fate.

N6-methyladenosine triggers renal fibrosis via enhancing translation and stability of ZEB2 mRNA.

In recent years, a surge in studies investigating N6-methyladenosine (m6A) modification in human diseases has occurred. However, the specific roles and mechanisms of m6A in kidney disease remain incompletely understood. This study revealed that m6A plays a positive role in regulating renal fibrosis (RF) by inducing epithelial-to-mesenchymal phenotypic transition (EMT) in renal tubular cells. Through comprehensive analyses, including m6A sequencing, RNA sequencing, and functional studies, we confirmed the pivotal involvement of zinc finger E-box binding homeobox 2 (ZEB2) in m6A-mediated RF and EMT. Notably, the m6A-modified coding sequence (CDS) of ZEB2 mRNA significantly enhances its translational elongation and mRNA stability by interacting with the YTHDF1/eEF-2 complex and IGF2BP3, respectively. Moreover, targeted demethylation of ZEB2 mRNA using the dm6ACRISPR system substantially decreases ZEB2 expression and disrupts the EMT process in renal tubular epithelial cells. In vivo and clinical data further support the positive influence of m6A/ZEB2 on RF progression. Our findings highlight the m6A-mediated regulation of RF through ZEB2, revealing a novel therapeutic target for RF treatment and enhancing our understanding of the impact of mRNA methylation on kidney disease.

Unraveling the independent role of METTL3 in m6A modification and tumor progression in esophageal squamous cell carcinoma.

METTL3 and METTL14 are traditionally posited to assemble the m6A methyltransferase complex in a stoichiometric 1:1 ratio, modulating mRNA fate via m6A modifications. Nevertheless, recent investigations reveal inconsistent expression levels and prognostic significance of METTL3 and METTL14 across various tumor types, challenging their consistent functional engagement in neoplastic contexts. A pan-cancer analysis leveraging The Cancer Genome Atlas (TCGA) data has identified pronounced disparities in the expression patterns, functional roles, and correlations with tumor burden between METTL3 and METTL14, particularly in esophageal squamous cell carcinoma (ESCC). Knockdown experiments of METTL3 in EC109 cells markedly suppress cell proliferation both in vitro and in vivo, whereas METTL14 knockdown shows a comparatively muted effect on proliferation and does not significantly alter METTL3 protein levels. mRNA sequencing indicates that METTL3 singularly governs the expression of 1615 genes, with only 776 genes co-regulated with METTL14. Additionally, immunofluorescence co-localization studies suggest discrepancies in cellular localization between METTL3 and METTL14. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analyses demonstrate that METTL3 uniquely associates with the Nop56p-linked pre-rRNA complex and mRNA splicing machinery, independent of METTL14. Preliminary bioinformatics and multi-omics investigations reveal that METTL3's autonomous role in modulating tumor cell proliferation and its involvement in mRNA splicing are potentially pivotal molecular mechanisms. Our study lays both experimental and theoretical groundwork for a deeper understanding of the m6A methyltransferase complex and the development of targeted tumor therapies focusing on METTL3.

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

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