ETS1 and RBPJ transcriptionally regulate METTL14 to suppress TGF-ß1-induced epithelial-mesenchymal transition in human bronchial epithelial cells.

Asthma is a chronic respiratory disease characterized by airway inflammation and remodeling. Epithelial-mesenchymal transition (EMT) of bronchial epithelial cells is considered to be a crucial player in asthma. Methyltransferase-like 14 (METTL14), an RNA methyltransferase, is implicated in multiple pathological processes, including EMT, cell proliferation and migration. However, the role of METTL14 in asthma remains uncertain. This research aimed to explore the biological functions of METTL14 in asthma and its underlying upstream mechanisms. METTL14 expression was down-regulated in asthmatic from three GEO datasets (GSE104468, GSE165934, and GSE74986). Consistent with this trend, METTL14 was decreased in the lung tissues of OVA-induced asthmatic mice and transforming growth factor-ß1 (TGF-ß1)-stimulated human bronchial epithelial cells (Beas-2B) in this study. Overexpression of METTL14 caused reduction in mesenchymal markers (FN1, N-cad, Col-1 and α-SMA) in TGF-ß1-treated cells, but caused increase in epithelial markers (E-cad), thus inhibiting EMT. Also, METTL14 suppressed the proliferation and migration ability of TGF-ß1-treated Beas-2B cells. Two transcription factors, ETS1 and RBPJ, could both bind to the promoter region of METTL14 and drive its expression. Elevating METTL14 expression could reversed EMT, cell proliferation and migration promoted by ETS1 or RBPJ deficiency. These results indicate that the ETS1/METTL14 and RBPJ/METTL14 transcription axes exhibit anti-EMT, anti-proliferation and anti-migration functions in TGF-ß1-induced bronchial epithelial cells, implying that METTL14 may be considered an alternative candidate target for the treatment of asthma.

METTL14 plays an oncogenic role in NSCLC by modulating ferroptosis and the m6A modification of GPX4.

CONTEXT: N6-methyladenosine (m6A) of RNA is involved in the progression of non-small cell lung cancer (NSCLC). OBJECTIVE: This study investigated the role of METTL14 in NSCLC and the mechanism. MATERIALS AND METHODS: Expression levels were assessed by quantitative real-time PCR and ELISA assays. Cells viability was assessed by cell counting kit-8. M6A methylation was analysed by methylated RNA immunoprecipitation (MeRIP), RIP, luciferase assay, and mRNA stability assay. RESULTS: The results showed that METTL14 was highly expressed in NSCLC tissues and cell lines. Knockdown of METTL14 inhibited the cell viability while induced ferroptosis of NSCLC cells. Mechanistically, METTL14 interacts with GPX4, mediates m6A modification of GPX4, enhances its mRNA stability, and upregulates its expression. In addition, IGF2BP1 recognises the m6A-methylated GPX4 and mediates the elevated mRNA stability. Moreover, GPX4 reversed the effects of METTL14 depletion. DISCUSSION AND CONCLUSION: The METTL14/GPX4 axis promotes NSCLC progression by inhibiting cell ferroptosis through the recognition of m6A modification mediated by IGF2BP1.

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

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

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.

METTL14 promotes lipid metabolism reprogramming and sustains nasopharyngeal carcinoma progression via enhancing m6A modification of ANKRD22 mRNA.

BACKGROUND: N6-methyladenosine (m6A) modification is essential for modulating RNA processing as well as expression, particularly in the context of malignant tumour progression. However, the exploration of m6A modification in nasopharyngeal carcinoma (NPC) remains very limited. METHODS: RNA m6A levels were analysed in NPC using m6A dot blot assay. The expression level of methyltransferase-like 14 (METTL14) within NPC tissues was analysed from public databases as well as RT-qPCR and immunohistochemistry. The influences on METTL14 expression on NPC proliferation and metastasis were explored via in vitro as well as in vivo functional assays. Targeted genes of METTL14 were screened using the m6A and gene expression profiling microarray data. Actinomycin D treatment and polysome analysis were used to detect the half-life and translational efficiency of ANKRD22. Flow cytometry, immunofluorescence and immunoprecipitation were used to validate the role of ANKRD22 on lipid metabolism in NPC cells. ChIP-qPCR analysis of H3K27AC signalling near the promoters of METTL14, GINS3, POLE2, PLEK2 and FERMT1 genes. RESULTS: We revealed METTL14, in NPC, correlating with poor patient prognosis. In vitro and in vivo assays indicated METTL14 actively promoted NPC cells proliferation and metastasis. METTL14 catalysed m6A modification on ANKRD22 messenger ribonucleic acid (mRNA), recognized by the reader IGF2BP2, leading to increased mRNA stability and higher translational efficiency. Moreover, ANKRD22, a metabolism-related protein on mitochondria, interacted with SLC25A1 to enhance citrate transport, elevating intracellular acetyl-CoA content. This dual impact of ANKRD22 promoted lipid metabolism reprogramming and cellular lipid synthesis while upregulating the expression of genes associated with the cell cycle (GINS3 and POLE2) and the cytoskeleton (PLEK2 and FERMT1) through heightened epigenetic histone acetylation levels in the nucleus. Intriguingly, our findings highlighted elevated ANKRD22-mediated histone H3 lysine 27 acetylation (H3K27AC) signals near the METTL14 promoter, which contributes to a positive feedback loop perpetuating malignant progression in NPC. CONCLUSIONS: The identified METTL14-ANKRD22-SLC25A1 axis emerges as a promising therapeutic target for NPC, and also these molecules may serve as novel diagnostic biomarkers.

METTL14 is Involved in TNF-α-Induced Inflammation in Colorectal Epithelial Cells via Autophagy Modulation.

Ulcerative colitis (UC) is a chronic and relapsing inflammatory bowel disease (IBD) characterized by colorectal inflammation. The N6-methyladenosine (m6A) modification of RNA regulates gene expression through the modulation of RNA metabolism, thus influencing various physiological and pathological processes. The aim of this study was to investigate the biological function of m6A methyltransferase METTL14 in colorectal epithelial cell inflammation. Bioinformatics analysis indicated that METTL14 expression was decreased in UC and was associated with disease severity and immune infiltration. We also noted a downregulation of METTL14 expression and a decrease in the total m6A RNA levels in TNF-α-stimulated Caco-2 cells. Moreover, METTL14 knockdown promoted inflammation and inhibited autophagy in TNF-α-stimulated Caco-2 cells, as indicated by the upregulation of NF-κB signaling and pro-inflammatory cytokine expression as well as LC3B protein downregulation. Treatment with the autophagy activator Torin-1 ameliorated the pro-inflammatory effects of METTL14 silencing. Furthermore, METTL14 knockdown significantly reduced the expression of ATG5. ATG5 overexpression could nullify the pro-inflammatory effect of METTL14 knockdown in TNF-α-stimulated Caco-2 cells. Mechanistically, METTL14 knockdown promoted ATG5 mRNA degradation, and luciferase analysis identified ATG5 as a target of m6A modification by METTL14. Taken together, silencing METTL14 promoted inflammation in Caco-2 cells via the downregulation of ATG5. Our findings revealed the importance of the m6A modification in colonic inflammation and autophagy, indicating that targeting METTL14 might be a potential therapeutic strategy for anti-inflammatory treatment in UC.

METTL14-mediated methylation of SLC25A3 mitigates mitochondrial damage in osteoblasts, leading to the improvement of osteoporosis.

PURPOSE: Osteoporosis is linked to impaired function of osteoblasts, and decreased expression of METTL14 may result in abnormal differentiation of these bone-forming cells. However, the specific impact of METTL14 on osteoblast differentiation and its underlying mechanisms are not yet fully understood. METHODS AND RESULTS: This study discovered a positive correlation between METTL14 expression and bone formation in specimens from osteoporosis patients and ovariectomized (OVX) mice. Additionally, METTL14 targeting of SLC25A3 contributed to the restoration of mitochondrial ROS levels and mitochondrial membrane potential in osteoblasts and promoted osteoblast differentiation. Moreover, in vivo experiments showed that METTL14 enhanced bone formation, and therapeutic introduction of METTL14 countered the decrease in bone formation in OVX mice. CONCLUSIONS: Overall, these findings emphasize the crucial role of the METTL14/SLC25A3 signaling axis in osteoblast activity, suggesting that this axis could be a potential target for improving osteoporosis.

Icariin upregulates methyltransferase-like 14-mediated prolyl 4-hydroxylase beta subunit m6A modification to promote osteogenic differentiation of bone marrow stem cells.

Prolyl 4-hydroxylase beta subunit (P4HB) plays a vital role in bone formation. This study intends to clarify the role of P4HB in the therapeutic effect of Icariin (ICA) on osteoporosis. Herein, in vivo and in vitro models were constructed by performing ovariectomy (OVX) in rats and inducing osteogenic differentiation in bone marrow stem cells (BMSCs), respectively. Hematoxylin and eosin staining and micro-computed tomography analysis were performed to evaluate osteoporosis in OVX rats. Alizarin Red staining, alkaline phosphatase staining, and the ALP activity test were employed to assess osteogenesis. m6A dot blotting and methylated RNA immunoprecipitation were used to determine m6A modification. We found that P4HB was downregulated in bone tissues of patients with osteoporosis and OVX rats. P4HB facilitated osteogenic differentiation of BMSCs. What's more, ICA upregulated P4HB expression, promoted osteogenic differentiation of BMSCs, and alleviated osteoporosis in OVX rats, which were reversed by knocking down P4HB. ICA enhanced the stability and m6A modification of P4HB. METTL14 mediated m6A modification of P4HB mRNA. In addition, METTL14 knockdown overturned the promotive effects of ICA on P4HB m6A level and BMSC osteogenic differentiation. To sum up, ICA elevated the METTL14-mediated m6A modification of P4HB to facilitate BMSC osteogenic differentiation.

METTL14 mediates nerve growth factor-stimulated testosterone synthesis in porcine theca cells.

Ovarian theca cells produce testosterone, which acts as a vital precursor substance for synthesizing estrogens during follicular development. Nerve growth factor (NGF) has been shown to participate in reproductive physiology, specifically to follicular development and ovulation. There is currently no available data on the impact of NGF on testosterone synthesis in porcine theca cells. Furthermore, m6A modification is the most common internal modification in eukaryotic mRNAs that are closely associated with female gametogenesis, follicle development, ovulation, and other related processes. It is also uncertain whether the three main enzymes associated with m6A, such as Writers, Erasers and Readers, play a role in this process. The present study, with an in vitro culture model, investigated the effect of NGF on testosterone synthesis in porcine theca cells and the role of Writers-METTL14 in this process. It was found that NGF activates the PI3K/AKT signaling pathway through METTL14, which regulates testosterone synthesis in porcine theca cells. This study will help to further elucidate the mechanisms by which NGF regulates follicular development and provide new therapeutic targets for ovary-related diseases in female animals.

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

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

METTL14 inhibits the malignant processes of gastric cancer cells by promoting N6-methyladenosine (m6A) methylation of TAF10.

N6-methyladenosine (m6A) methylation mediates cancer development by regulating cell proliferation and metastasis. This study aimed to identify whether methyltransferase 14 (METTL14) affects gastric cancer (GC) cellular functions and its underlying mechanism. METTL14 and TATA-box binding protein associated factor 10 (TAF10) levels were examined using quantitative real-time PCR, immunohistochemical assay, and Western blot. Biological functions were assessed using cell counting kit-8, colony formation, and transwell assays. The interaction between METTL14 and TAF10 was analyzed using RNA immunoprecipitation, methylated RNA immunoprecipitation, and luciferase reporter assay. A xenograft tumor mouse model was established to assess the role of METTL14 in vivo. The results suggested that METTL14 was low expressed and TAF10 was highly expressed in GC tissues and cells. METTL14 overexpression inhibited GC cell viability, colony, migration, and invasion. TAF10 was predicted and confirmed to be negatively related to METTL14. METTL14 promoted m6A methylation of TAF10 and inhibited TAF10 stability. Moreover, TAF10 counteracted the cellular behaviors regulated by METTL14. Overexpression of METTL14 inhibited tumor growth and histopathology. In conclusion, METTL14 inhibits GC progression by attenuating GC cell proliferation, migration, and invasion. Mechanistically, METTL14 promoted m6A methylation of TAF10, suppressed the stability of TAF10, and thus downregulated the TAF10 levels, These results provide a new insight into GC therapy.

METTL14 decreases FTH1 mRNA stability via m6A methylation to promote sorafenib-induced ferroptosis of cervical cancer.

Cervical cancer (CC) is a prevalent malignancy among women worldwide. This study was designed to investigate the role of METTL14 in sorafenib-induced ferroptosis in CC. METTL14 expression and m6A methylation were determined in CC tissues, followed by analyzes correlating these factors with clinical features. Subsequently, METTL14 was knocked down in CC cell lines, and the effects on cell proliferation, mitochondrial morphology and ferroptosis were assessed using CCK-8, microscopy, and markers associated with ferroptosis, respectively. The regulatory relationship between METTL14 and FTH1 was verified using qRT-PCR and luciferase reporter assays. The functional significance of this interaction was further investigated both in vitro and in vivo by co-transfecting cells with overexpression vectors or shRNAs targeting METTL14 and FTH1 after sorafenib treatment. METTL14 expression and m6A methylation were significantly reduced in CC tissues, and lower METTL14 expression levels were associated with a poorer CC patients' prognosis. Notably, METTL14 expression increased during sorafenib-induced ferroptosis, and METTL14 knockdown attenuated the ferroptotic response induced by sorafenib in CC cells. FTH1 was identified as a direct target of METTL14, with METTL14 overexpression leading to increased m6A methylation of FTH1 mRNA, resulting in reduced stability and expression of FTH1 in CC. Furthermore, FTH1 overexpression or treatment with LY294002 partially counteracted the promotion of sorafenib-induced ferroptosis by METTL14. In vivo xenograft experiments demonstrated that inhibiting METTL14 reduced the anticancer effects of sorafenib, whereas suppression of FTH1 significantly enhanced sorafenib-induced ferroptosis and increased its anticancer efficacy. METTL14 reduces FTH1 mRNA stability through m6A methylation, thereby enhancing sorafenib-induced ferroptosis, which contributes to suppressing CC progression via the PI3K/Akt signaling pathway.

CircUGGT2 downregulation by METTL14-dependent m6A modification suppresses gastric cancer progression and cisplatin resistance through interaction with miR-186-3p/MAP3K9 axis.

Chemoresistance is a major therapeutic challenge in advanced gastric cancer (GC). N6-methyladenosine (m6A) RNA modification has been shown to play fundamental roles in cancer progression. However, the underlying mechanisms by which m6A modification of circRNAs contributes to GC and chemoresistance remain unknown. We found that hsa_circ_0030632 (circUGGT2) was a predominant m6A target of METTL14, and METTL14 knockdown (KD) reduced circUGGT2 m6A levels but increased its mRNA levels. The expression of circUGGT2 was markedly increased in cisplatin (DDP)-resistant GC cells. CircUGGT2 KD impaired cell growth, metastasis and DDP-resistance in vitro and in vivo, but circUGGT2 overexpression prompted these effects. Furthermore, circUGGT2 was validated to sponge miR-186-3p and upregulate MAP3K9 and could abolish METTL14-caused miR-186-3p upregulation and MAP3K9 downregulation in GC cells. circUGGT2 negatively correlated with miR-186-3p expression and harbored a poor prognosis in patients with GC. Our findings unveil that METTL14-dependent m6A modification of circUGGT2 inhibits GC progression and DDP resistance by regulating miR-186-3p/MAP3K9 axis.

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

BACKGROUND: N6-methyladenosine (m6A) modification plays an important role in lung cancer. However, methyltransferase-like 14 (METTL14), which serves as the main component of the m6A complex, has been less reported to be involved in the immune microenvironment of lung cancer. This study aimed to analyze the relationship between METTL14 and the immune checkpoint inhibitor programmed death receptor 1 (PD-1) in lung cancer. METHODS: CCK-8, colony formation, transwell, wound healing, and flow cytometry assays were performed to explore the role of METTL14 in lung cancer progression in vitro. Furthermore, syngeneic model mice were treated with sh-METTL14 andan anti-PD-1 antibody to observe the effect of METTL14 on immunotherapy. Flow cytometry and immunohistochemical (IHC) staining were used to detect CD8 expression. RIP and MeRIP were performed to assess the relationship between METTL14 and HSD17B6. LLC cells and activated mouse PBMCs were cocultured in vitro to mimic immune cell infiltration in the tumor microenvironment. ELISA was used to detect IFN-γ and TNF-α levels. RESULTS: The online database GEPIA showed that high METTL14 expression indicated a poor prognosis in patients with lung cancer. In vitro assays suggested that METTL14 knockdown suppressed lung cancer progression. In vivo assays revealed that METTL14 knockdown inhibited tumor growth and enhanced the response to PD-1 immunotherapy. Furthermore, METTL14 knockdown enhanced CD8+T-cell activation and infiltration. More importantly, METTL14 knockdown increased the stability of HSD17B6 mRNA by reducing its m6A methylation. In addition, HSD17B6 overexpression promoted the activation of CD8+ T cells. CONCLUSION: The disruption of METTL14 contributed to CD8+T-cell activation and the immunotherapy response to PD-1 via m6A modification of HSD17B6, thereby suppressing lung cancer progression.

METTL14/YTHDC1-Mediated m6A Modification in Hippocampus Improves Pentylenetetrazol-Induced Acute Seizures.

Epilepsy is a common neurological disorder which can cause significant morbidity and mortality. N6-methyladenosine (m6A), the most common chemical epigenetic modification among mRNA post-transcriptional modifications, implicated in various physiological and pathological processes, but its role in epilepsy is still unknown. Here, we provide strong evidences in support of an association of m6A and its regulatory proteins with epilepsy. Our results indicated that the level of m6A was declined significantly in the dentate gyrus (DG) of hippocampus of pentylenetetrazol (PTZ)-induced seizure mice. Both the seizure-like behaviors and the excessive activation of DG area neuron were significantly mitigated after the administration of m6A agonist betaine. Mechanically, we found that both the m6A methyltransferase METTL14 and recognition protein YTHDC1 were decreased by PTZ stimulation, which might contribute to the reduced m6A level. Additionally, DG-specific over-expression of METTL14 or YTHDC1 by lentivirus injection could significantly ameliorate seizure-like behaviors and prevent the excessive activation of neuron in epilepsy mice induced by PTZ injection, which might be due to the normalized m6A level. Together, this study identified that METTL14/YTHDC1-mediated m6A modification could participate in seizure-like behaviors, which might provide m6A regulation as a potential and novel therapeutic strategy for epilepsy.

CHOP regulated by METTL14-m6A affects cell cycle arrest and regorafenib sensitivity in HCC cells.

BACKGROUND: Regorafenib, a multi-targeted kinase inhibitor, has been used in the treatment of Hepatocellular carcinoma (HCC). The purpose of this study is to investigate the mechanism of Regorafenib in HCC. METHODS: Regorafenib's impact on the sensitivity of HCC cells was assessed using CCK8. Differential gene expression analysis was performed by conducting mRNA sequencing after treatment with Regorafenib. The m6A methylation status of CHOP and differential expression of m6A methylation-related proteins were assessed by RIP and Western Blot. To explore the molecular mechanisms involved in the therapeutic effects of Regorafenib in HCC and the impact of METTL14 and CHOP on Regorafenib treatment, we employed shRNA/overexpression approaches to transfect METTL14 and CHOP genes, as well as conducted in vivo experiments. RESULTS: Treatment with Regorafenib led to a notable decrease in viability and proliferation of SK-Hep-1 and HCC-LM3 cells. The expression level of CHOP was upregulated after Regorafenib intervention, and CHOP underwent m6A methylation. Among the m6A methylation-related proteins, METTL14 exhibited the most significant downregulation. Mechanistic studies revealed that Regorafenib regulated the cell cycle arrest in HCC through METTL14-mediated modulation of CHOP, and the METTL14/CHOP axis affected the sensitivity of HCC to Regorafenib. In vivo, CHOP enhanced the anticancer effect of Regorafenib. CONCLUSION: The inhibition of HCC development by Regorafenib is attributed to its modulation of m6A expression of CHOP, mediated by METTL14, and the METTL14/CHOP axis enhances the sensitivity of HCC to Regorafenib. These findings provide insights into the treatment of HCC and the issue of drug resistance to Regorafenib.

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

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

Aberrant METTL14 gene expression contributes to malignant transformation of benzene-exposed myeloid cells.

Benzene is a known contributor to human leukaemia through its toxic effects on bone marrow cells, and epigenetic modification is believed to be a potential mechanism underlying benzene pathogenesis. However, the specific roles of N6-methyladenosine (m6A), a newly discovered RNA post-transcriptional modification, in benzene-induced hematotoxicity remain unclear. In this study, we identified self-renewing malignant proliferating cells in the bone marrow of benzene-exposed mice through in vivo bone marrow transplantation experiments and Competitive Repopulation Assay. Subsequent analysis using whole transcriptome sequencing and RNA m6A methylation sequencing revealed a significant upregulation of RNA m6A modification levels in the benzene-exposed group. Moreover, RNA methyltransferase METTL14, known as a pivotal player in m6A modification, was found to be aberrantly overexpressed in Lin-Sca-1+c-Kit+ (LSK) cells of benzene-exposed mice. Further analysis based on the GEO database showed a positive correlation between the expression of METTL14, mTOR, and GFI and benzene exposure dose. In vitro cellular experiments, employing experiments such as western blot, q-PCR, m6A RIP, and CLIP, validated the regulatory role of METTL14 on mTOR and GFI1. Mechanistically, continuous damage inflicted by benzene exposure on bone marrow cells led to the overexpression of METTL14 in LSK cells, which, in turn, increased m6A modification on the target genes' (mTOR and GFI1) RNA. This upregulation of target gene expression activated signalling pathways such as mTOR-AKT, ultimately resulting in malignant proliferation of bone marrow cells. In conclusion, this study offers insights into potential early targets for benzene-induced haematologic malignant diseases and provides novel perspectives for more targeted preventive and therapeutic strategies.

N6-methyladenosine methylation on FSCN1 mediated by METTL14/IGF2BP3 contributes to human papillomavirus type 16-infected cervical squamous cell carcinoma.

Human papillomavirus (HPV) infection has been reported to be associated with N6-methyladenosine (m6A) modification in cancers. However, the underlying mechanism by which m6A methylation participates in HPV-related cervical squamous cell carcinoma (CSCC) remains largely unclear. In this study, we observed that m6A regulators methyltransferase like protein (METTL14) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) were upregulated in HPV-positive CSCC tissues and cell lines, and their high expression predicted poor prognosis for HPV-infected CSCC patients. Cellular functional experiments verified that HPV16 oncogenes E6/E7 upregulated the expression of METTL14 and IGF2BP3 to promote cell proliferation and epithelial mesenchymal transition of CSCC cells. Next, we found that E6/E7 stabilized fascin actin-bundling protein 1 (FSCN1) mRNA and elevated FSCN1 expression in CSCC cells through upregulating METTL14/IGF2BP3-mediated m6A modification, and FSCN1 expression was also validated to be positively associated with worse outcomes of HPV-positive CSCC patients. Finally, HPV16-positive CSCC cell lines SiHa and CaSki were transfected with knockdown vector for E6/E7 or METTL14/IGF2BP3 and overexpressing vector for FSCN1, and functional verification experiments were performed through using MTT assay, flow cytometry, wound healing assay and tumour formation assay. Results indicated that knockdown of E6/E7 or METTL14/IGF2BP3 suppressed cell proliferation, migration and tumorigenesis, and accelerated cell apoptosis of HPV-positive CSCC cells. Their tumour-suppressive effects were abolished through overexpressing FSCN1. Overall, HPV E6/E7 advanced CSCC development through upregulating METTL14/IGF2BP3-mediated FSCN1 m6A modification.

Epigenetic activation of METTL14 promotes docetaxel resistance in prostate cancer by promoting pri-microRNA-129 maturation.

The development of resistance to Docetaxel (DTX) compromises its therapeutic efficacy and worsens the prognosis of prostate cancer (PCa), while the underlying regulatory mechanism remains poorly understood. In this study, METTL14 was found to be upregulated in DTX-resistant PCa cells and PCa tissues exhibiting progressive disease during DTX therapy. Furthermore, overexpression of METTL14 promoted the development of resistance to DTX in both in vitro and in vivo. Interestingly, it was observed that the hypermethylation of the E2F1 targeting site within DTX-resistant PCa cells hindered the binding ability of E2F1 to the promoter region of METTL14, thereby augmenting its transcriptional activity. Consequently, this elevated expression level of METTL14 facilitated m6A-dependent processing of pri-miR-129 and subsequently led to an increase in miR-129-5p expression. Our study highlights the crucial role of the E2F1-METTL14-miR-129-5p axis in modulating DTX resistance in PCa, underscoring METTL14 as a promising therapeutic target for DTX-resistant PCa patients.