Gubi decoction mitigates knee osteoarthritis via promoting chondrocyte autophagy through METTL3-mediated ATG7 m6A methylation.

Knee osteoarthritis (KOA) is a chronic joint disease that significantly affects the health of the elderly. As an herbal remedy, Gubi decoction (GBD) has been traditionally used for the treatment of osteoarthritis-related syndromes. However, the anti-KOA efficacy and mechanism of GBD remain unclear. This study aimed to experimentally investigate the anti-KOA efficacy and the underlying mechanism of GBD. The medial meniscus (DMM) mice model and IL-1ß-stimulated chondrocytes were, respectively, constructed as in vivo and in vitro models of KOA to evaluate the osteoprotective effect and molecular mechanism of GBD. The UPLC-MS/MS analysis showed that GBD mainly contained pinoresinol diglucoside, rehmannioside D, hesperidin, liquiritin, baohuoside I, glycyrrhizic acid, kaempferol and tangeretin. Animal experiment showed that GBD could alleviate articular cartilage destruction and recover histopathological alterations in DMM mice. In addition, GBD inhibited chondrocyte apoptosis and restored DMM-induced dysregulated autophagy evidenced by the upregulation of ATG7 and LC3 II/LC3 I but decreased P62 level. Mechanistically, METTL3-mediated m6A modification decreased the expression of ATG7 in DMM mice, as it could be significantly attenuated by GBD. METTL3 overexpression significantly counteracted the protective effect of GBD on chondrocyte autophagy. Further research showed that GBD promoted proteasome-mediated ubiquitination degradation of METLL3. Our findings suggest that GBD could act as a protective agent against KOA. The protective effect of GBD may result from its promotion on chondrocyte autophagy by suppressing METTL3-dependent ATG7 m6A methylation.

METTL3-Regulated lncRNA SNHG7 Drives MNNG-Induced Epithelial-Mesenchymal Transition in Gastric Precancerous Lesions.

As a representative item of chemical carcinogen, MNNG is closely associated with the onset of gastric cancer (GC), where N6-methyladonosine (m6A) RNA methylation is recognized as a critical epigenetic event. In our previous study, we found that the m6A modification by methyltransferase METTL3 was up-regulated in MNNG-exposed malignant GES-1 cells (MC cells) compared to control cells in vitro, and long non-coding RNA SNHG7 as a downstream target of the METTL3. However, the functional role of METTL3 in mediating the SNHG7 axis in MNNG-induced GC remains unclear. In the present study, we continuously investigate the functional role of METTL3 in mediating the SNHG7 axis in MNNG-induced GC. RIP-PCR and m6A-IP-qPCR were used to examine the molecular mechanism underlying the METTL3/m6A/SNHG7 axis in MNNG-induced GC. A METTL3 knockout mice model was constructed and exposed by MNNG. Western blot analysis, IHC analysis, and RT-qPCR were used to measure the expression of METTL3, SNHG7, and EMT markers. In this study, we demonstrated that in MNNG-induced GC tumorigenesis, the m6A modification regulator METTL3 facilitates cellular EMT and biological functions through the m6A/SNHG7 axis using in vitro and in vivo models. In conclusion, our study provides novel insights into critical epigenetic molecular events vital to MNNG-induced gastric carcinogenesis. These findings suggest the potential therapeutic targets of METTL3 for GC treatment.

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.

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.

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.

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.

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.

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

Background: 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. Objective: This study aims to elucidate the mechanisms by which METTL3 contributes to kanamycin-induced ototoxicity. Methods and Results: 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. Conclusion: 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.

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 in cancer-associated fibroblasts-derived exosomes promotes the proliferation and metastasis and suppresses ferroptosis in colorectal cancer by eliciting ACSL3 m6A modification.

BACKGROUND: Cancer-associated fibroblasts (CAFs) have been reported that can affect cancer cell proliferation, metastasis, ferroptosis, and immune escape. METTL3-mediated N6-methyladenine (m6A) modification is involved in the tumorigenesis of colorectal cancer (CRC). Herein, we investigated whether METTL3-dependent m6A in CAFs-derived exosomes (exo) affected CRC progression. METHODS: qRT-PCR and western blotting analyses detected levels of mRNAs and proteins. Cell proliferation and metastasis were evaluated using MTT, colony formation, transwell, and wound healing assays, respectively. Cell ferroptosis was assessed by detecting cell viability and the levels of Fe+, reactive oxygen species, and glutathione after erastin treatment. Exosomes were isolated from CAFs by ultracentrifugation. The m6A modification profile was determined by methylated RNA immunoprecipitation assay and the interaction between METTL3 and ACSL3 (acyl-CoA synthetase 3) was verified using dual-luciferase reporter assay. Animal models were established for in vivo analysis. RESULTS: CAFs promoted CRC cell proliferation and metastasis, and suppressed cell ferroptosis. METTL3 was enriched in CAFs and was packaged into exosomes. The m6A modification and METTL3 expression were increased in CRC samples. Knockdown of METTL3 in CAFs-exo suppressed CRC cell proliferation and metastasis, and induced cell ferroptosis. Mechanistically, METTL3 induced ACSL3 m6A modification and stabilized its expression. The anticancer effects mediated by METTL3-silenced CAFs-exo could be rescued by ACSL3 overexpression. Moreover, in vivo assay also showed that CAFs-exo with decreased METTL3 could hinder CRC growth and metastasis in mice models. CONCLUSION: CAFs promoted the proliferation and metastasis, and restrained the ferroptosis in CRC by exosomal METTL3-elicited ACSL3 m6A modification.

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.

METTL3 mediates m6A modification of hsa_circ_0072380 to regulate the progression of gestational diabetes mellitus.

BACKGROUND: m6A modification plays a vital role in gestational diabetes mellitus (GDM) progression. However, the role of METTL3 and differential m6A-modified circRNAs in GDMstayto be investigated. METHODS: Placental tissue samples from GDM patients and normal controls (NC) were collected to measure changes in m6A modification levels. MeRIP-seq on placental tissue was performed to detect differential m6A-modified circRNAs.High glucose (HG)-treated JEG3 cells were used to establish the GDM cell model. Differentially expressed circRNAs levels in GDM and NC groups was measured by qRT-PCR. We knocked down METTL3 to study its function. Additionally, we conducted functional recovery experiments. Dot blot assay was utilized to assess changes in m6A levels. MeRIP-qPCR was performed to evaluate the effect of knocking down METTL3 on m6A modification of hsa_circ_0072380 in JEG3 cells. RESULTS: Compared with NC group, GDM group exhibited increased levels of m6A modification and METTL3 expression. Differences in m6A modification of circRNAs exist between the GDM and NC groups. Hsa_circ_0000994, hsa_circ_0058733, and hsa_circ_0072380 were significantly down-regulated in GDM group while hsa_circ_0036376, hsa_circ_0000471, and hsa_circ_0001173 showed no significant differences between two groups. HG treatment promoted METTL3 expression and m6A level of JEG3 cells, and inhibited cell proliferation, migration, and invasion abilities. Knocking down METTL3 reversed these effects. After HG treatment, hsa_circ_0072380 was significantly down-regulated. Knocking down METTL3 led to up-regulation of hsa_circ_0072380, while knocking down hsa_circ_0072380 restored the function of SiMETTL3. Additionally, knocking down METTL3 significantly reduced m6A modification of hsa_circ_0072380. CONCLUSION: METTL3 mediated m6A modification of hsa_circ_0072380 to regulate GDM progression.

METTL3 -mediated m6A modification of circ_0000620 regulates cisplatin sensitivity and apoptosis in lung adenocarcinoma via the MiR-216b-5p/KRAS axis.

Circular RNAs (circRNAs) are stable non-coding RNAs characterized by the absence of the conventional 5' cap and 3' polyadenylated tail structure. Its involvement in various aspects of cancers underscores its significance in oncology. Elevated expression of circ_0000620 was observed in both lung adenocarcinoma (LUAD) tissues and cell lines. In vitro, experiments demonstrated that the downregulation of circ_0000620 increased cisplatin sensitivity and promoted cell apoptosis while suppressing malignant characteristics such as cell migration and proliferation. Further investigation into the mechanism underlying the increased expression of circ_0000620 revealed that Methyltransferase 3, N6-Adenosine-Methyltransferase Complex Catalytic Subunit (METTL3) mediates the m6A methylation modification of circ_0000620, thereby promoting its stability and expression. Furthermore, circ_0000620 modulates the miR-216b-5p/KRAS axis to influence apoptosis and cisplatin sensitivity in both A549 and H1299 cell lines. These findings were corroborated by in vivo nude mouse experiments, which showed that knockdown of circ_0000620 inhibited tumor growth and proliferation. In summary, METTL3 plays a role in regulating the stability of circ_0000620 expression, and circ_0000620 exerts its effects on LUAD apoptosis and cisplatin sensitivity through the miR-216b-5p/KRAS signaling pathway.

Single-cell sequencing reveals that specnuezhenide protects against osteoporosis via activation of METTL3 in LEPR+ BMSCs.

BACKGROUND: Osteoporosis (OP) has garnered significant attention due to its substantial morbidity and mortality rates, imposing considerable health burdens on societies worldwide. However, the molecular mechanisms underlying osteoporosis pathogenesis remain largely elusive, and the available therapeutic interventions are limited. Therefore, there is an urgent need for innovative strategies in the treatment of osteoporosis. PURPOSE: The primary objective of this study was to elucidate the molecular mechanisms underlying osteoporosis pathogenesis using single-cell RNA sequencing (scRNA-seq), thereby proposing novel therapeutic agents. METHODS: The mice osteoporosis model was established through bilateral ovariectomy. Micro-computed tomography (µCT) and hematoxylin and eosin (H&E) staining were employed to assess the pathogenesis of osteoporosis. scRNA-seq was utilized to identify and analyze distinct molecular mechanisms and sub-clusters. Gradient dilution analysis was used to obtain specific sub-clusters, which were further validated by immunofluorescence staining and flow cytometry analysis. Molecular docking and cellular thermal shift assay (CETSA) were applied for screening potential agents in the TCMSPs database. Alkaline phosphatase (ALP) activity and alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Osteogenic organoids analysis was employed to assess the proliferation and sphere-forming ability of BMSCs. Quantitative real-time PCR (qRT-PCR) and western blot analysis were conducted to investigate signaling pathways. Wound healing assay and tube formation analysis were employed to evaluate the angiogenesis of endothelial cells. RESULTS: The scRNA-seq analysis revealed the crucial role of LEPR+ BMSCs in the pathogenesis of osteoporosis, which was confirmed by immunofluorescence staining of the epiphysis. Subsequently, the LEPR+ BMSCs were obtained by gradient dilution analysis and identified by immunofluorescence staining and flow cytometry. Accordingly, specnuezhenide (Spe) was screened and identified as a potential compound targeting METTL3 from the TCMSPs database. Spe promoted bone formation as evidenced by µ-CT, and H&E analysis. Additionally, Spe enhanced the osteogenic capacity of LEPR+ BMSCs through ALP and ARS assay. Notably, METTL3 pharmacological inhibitors S-Adenosylhomocysteine (SAH) attenuated the aforementioned osteo-protective effects of Spe. Particularly, Spe enhanced the LEPR+ BMSCs-dependent angiogenesis through the secretion of SLIT3, which was abolished by SAH in LEPR+ BMSCs. CONCLUSION: Collectively, these findings suggest that Spe could enhance the osteogenic potential of LEPR+ BMSCs and promote LEPR+ BMSCs-dependent angiogenesis by activating METTL3 in LEPR+ BMSCs, indicating its potential as an ideal therapeutic agent for clinical treatment of osteoporosis.

SRF Facilitates Transcriptional Inhibition of Gem Expression by m6A Methyltransferase METTL3 to Suppress Neuronal Damage in Epilepsy.

A bioinformatics analysis was conducted to screen for relevant expression datasets of the transcription factor SRF knockout mice. The aim was to investigate the relationship between SRF and m6A-related genes, predict how SRF regulates the m6A modification of GEM genes mediated by METTL3, and explore potential molecular mechanisms associated with neurotrauma. Disease gene databases such as GeneCards, DisGeNET, and Phenolyzer, and transcription factor databases TFDB and TRRUST, were used to obtain epilepsy-related genes and transcription factors. The intersection was then selected. Expression data of SRF knockout epilepsy mice were obtained from the GEO database and used to filter differentially expressed genes. Important module genes related to the disease were selected through WGCNA co-expression analysis. The intersection between these genes and the differentially expressed genes was performed, followed by PPI network analysis and GO/KEGG enrichment analysis. Furthermore, the core genes were selected using the cytoHubba plugin of the Cytoscape software. Differential expression analysis was performed on m6A-related factors in the GEO dataset, and the relationship between SRF and m6A-related factors and core genes was analyzed. The m6A binding sites of SRF with the METTL3 promoter and target gene Gem were predicted using the AnimalTFDB and SRAMP websites, respectively. We found that the transcription factor SRF may be a key gene in epilepsy during neuronal development. Further WGCNA analysis showed that 129 module genes were associated with SRF knockout epilepsy, and these differentially expressed genes were mainly enriched in the neuroactive ligand-receptor interaction pathway. The final results indicate that knocking out SRF may inhibit the transcription of METTL3, thereby inhibiting the m6A modification of Gem and leading to upregulation of Gem expression, thereby playing an important role in neuronal damage. Knocking out the SRF gene may inhibit the transcription of m6A methyltransferase METTL3, thereby inhibiting the m6A modification of GEM genes mediated by METTL3, promoting GEM gene expression, and leading to the occurrence of epilepsy-related neuron injury. Further investigation revealed that SRF overexpression can potentially enhance the transcription of METTL3, thus promoting m6A modification of GEM, resulting in downregulation of GEM expression. This process regulates oxidative stress in epileptic mouse neurons, suppresses inflammatory responses, and mitigates associated damage. Additionally, an in vitro neuronal epileptic model was established, and experimental techniques such as qRT-PCR and WB were employed to assess the expression of SRF, METTL3, and GEM in hippocampal tissues and neurons. The experimental results were consistent with our predictions, demonstrating that overexpression of SRF can inhibit the development of epilepsy-related neuronal damage. This study reveals that knockout of the SRF gene may suppress the transcription of m6A methyltransferase METTL3, thereby inhibiting m6A modification of the GEM gene mediated by METTL3 and subsequently promoting the expression of the GEM gene, leading to the occurrence of epilepsy-related neuronal damage.