METTL3-mediated m6A Research Articles

The "m6A writer" METTL3 and the "m6A reader" IGF2BP2 regulate cutaneous T-cell lymphomas progression via CDKN2A.

It has been established that Cutaneous T-Cell lymphomas (CTCL) are caused by the monoclonal proliferation of T lymphocytes in the skin. This heterogeneous group of diseases represents a significant source of distress to patients since the diagnosis and treatment are often challenging. As one of the most abundant internal modifications in mRNA in higher eukaryotes, N6-methyladenosine (m6A) is widely recognized to affect the development and progression of cancers. However, knowledge on the involvement of m6A in CTCL is still limited. In this work, we revealed the role of METTL3-mediated m6A modification in CTCL progression. ELISA, western blot, and qRT-PCR assays demonstrated that METTL3 was significantly downregulated in CTCL cells both in vivo and in vitro. CCK-8, EdU, flow cytometry, and transwell assays showed that the decline in METTL3 levels was responsible for CTCL cell proliferation and migration. Furthermore, using small interfering RNAs against METTL3 and the RIP assay, we showed that CDKN2A was a key regulator during this process in vitro and in vivo, and insufficient methylation modification blocked the interaction between CDKN2A and m6A reader IGF2BP2, resulting in mRNA degradation. To the best of our knowledge, this is the first study to depict the role of m6A in CTCL development and provide potential bio-targets for therapy.

Molecular mechanism of m6A methylation of circDLC1 mediated by RNA methyltransferase METTL3 in the malignant proliferation of glioma cells

Glioma is an intracranial malignant tumor and remains largely incurable. Circular RNAs are prominent modulators in glioma progression. This study investigated the function of circular RNA DLC1 (circDLC1) in the malignant proliferation of glioma cells. circDLC1 expression in glioma tissues and cells was determined using RT-qPCR. The effect of circDLC1 on the malignant proliferation of glioma cells was analyzed using CCK-8, colony formation, and EdU staining assays. METTL3, miR-671-5p, and CTNNBIP1 expressions were determined. N6 methyladenosine (m6A) level of circDLC1 was analyzed using MeRIP. The binding relationship between miR-671-5p and circDLC1 or CTNNBIP1 was verified using RNA pull-down and dual-luciferase assays. A xenograft tumor model was established in nude mice to verify the effect of METTL3-mediated circDLC1 on glioma in vivo. circDLC1 was poorly expressed in glioma. circDLC1 overexpression suppressed glioma cell proliferation. Mechanically, METTL3-mediated m6A modification enhanced circDLC1 stability and upregulated circDLC1 expression in glioma. circDLC1 upregulated CTNNBIP1 transcription by competitively binding to miR-671-5p. METTL3 overexpression repressed the malignant proliferation of glioma via circDLC1/miR-671-5p/CTNNBIP1 in vivo. Collectively, METTL3-mediated m6A modification upregulated circDLC1 expression, and circDLC1 promoted CTNNBIP1 transcription by sponging miR-671-5p, thus repressing the malignant proliferation of glioma.

Exenatide ameliorates hydrogen peroxide-induced pancreatic β-cell apoptosis through regulation of METTL3-mediated m6A methylation

Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, is a commonly used hypoglycemic agent in clinical practice; it inhibits reactive oxygen species-induced pancreatic β-cell apoptosis. N6-methyladenosine (m6A) is produced by the methylation of RNA N6 residues and has recently been shown to play a crucial role in the regulation of islet β-cell growth and development. However, the involvement of m6A methylation in the β-cell protective effects of exenatide has not been clarified. In this study, the m6A-methylated RNA content and methyltransferase-like 3 (METTL3) expression levels in NIT-1 cells and primary mouse islets were found to significantly decrease following treatment with hydrogen peroxide (H2O2). Treatment with exenatide induced an increase in m6A content and METTL3 expression in the H2O2-treated NIT-1 cells and islets. Moreover, METTL3 silencing resulted in NIT-1 cell apoptosis under normal culture conditions. METTL3 upregulation significantly ameliorated H2O2-induced apoptosis in NIT-1 cells and primary islets. Furthermore, the anti-apoptotic effects of exenatide were obviously reversed by METTL3 knockdown. In conclusion, these findings suggest that exenatide elicits its anti-apoptotic effects in pancreatic β-cells by promoting m6A methylation through the upregulation METTL3 expression.

METTL3 stabilizes HDAC5 mRNA in an m6A-dependent manner to facilitate malignant proliferation of osteosarcoma cells

Osteosarcoma (OS) is a prevalent primary bone sarcoma. Methyltransferase-like 3 (METTL3) is dysregulated in human malignancies. This study explored the mechanism of METTL3 in OS cell proliferation. Our results demonstrated that METTL3 was highly expressed in OS, and correlated with the tumor size, clinical stage, and distant metastasis of OS patients. Higher METTL3 expression indicated poorer prognosis. METTL3 silencing inhibited the malignant proliferation of OS cells, while METTL3 overexpression led to an opposite trend. METTL3 upregulated histone deacetylase 5 (HDAC5) expression in OS cells by increasing the m6A level. HDAC5 reduced the enrichment of H3K9/K14ac on miR-142 promoter, thus suppressing miR-142-5p expression and upregulating armadillo-repeat-containing 8 (ARMC8) level. HDAC5 overexpression or miR-142-5p silencing attenuated the inhibitory effect of METTL3 silencing on OS cell proliferation. Xenograft tumor experiment in nude mice confirmed that METTL3 silencing repressed OS cell proliferation in vivo via the HDAC5/miR-142-5p/ARMC8 axis. Collectively, METTL3-mediated m6A modification facilitated OS cell proliferation via the HDAC5/miR-142-5p/ARMC8 axis.

METTL3-mediated m6A modification of STEAP2 mRNA inhibits papillary thyroid cancer progress by blocking the Hedgehog signaling pathway and epithelial-to-mesenchymal transition

Papillary thyroid cancer (PTC) is a common endocrine system malignancy all over the world. Aberrant expression of six transmembrane epithelial antigen of the prostate 2 (STEAP2) has been functionally associated with cancer progression in many cancers. Nevertheless, its biological function in PTC is still unclear. Here, we found that PTC tissues had preferentially downregulated STEAP2 as compared with noncancerous tissues. Low STEAP2 expression correlated with aggressive clinicopathological characteristics and dismal prognosis in patients with PTC. We performed gain- and loss-of-function experiments, including cell proliferation assay (Cell Counting Kit-8 assay), EdU (5-ethynyl-2′-deoxyuridine) and colony formation assays, transwell migration, and invasion assays, and constructed a nude mouse xenograft tumor model. The results demonstrated that STEAP2 overexpression inhibited PTC cell proliferation, migration, and invasion in vitro and inhibited lung metastasis and tumorigenicity in vivo. Conversely, silencing STEAP2 yielded the opposite results in vitro. Mechanistically, bioinformatics analysis combined with validation experiments identified STEAP2 as the downstream target of methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification. METTL3 stabilized STEAP2 mRNA and regulated STEAP2 expression positively in an m6A-dependent manner. We also showed that m6A-mediated STEAP2 mRNA translation initiation relied on a pathway dependent on the m6A reader protein YTHDF1. Rescue experiments revealed that silencing STEAP2 partially rescued the tumor-suppressive phenotype induced by METTL3 overexpression. Lastly, we verified that the METTL3–STEAP2 axis functions as an inhibitor in PTC by suppressing epithelial–mesenchymal transition and the Hedgehog signaling pathway. Taken together, these findings strongly suggest that METTL3-mediated STEAP2 m6A modification plays a critical tumor-suppressive role in PTC progression. The METTL3–STEAP2 axis may be a potential therapeutic molecular target against PTC.

Mechanism of METTL3-mediated m6A modification in depression-induced cognitive deficits.

Depressive disorder (DD) is associated with N6-methyladenosine (m6A) hypermethylation. This study sought to explore the molecular mechanism of Methyltransferase-like 3 (METTL3) in cognitive deficits of chronic unpredictable mild stress (CUMS)-treated rats and provide novel targets for DD treatment. A DD rat model was established via CUMS treatment. Cognitive deficits were assessed via body weighing and behavioral tests. METTL3, microRNA (miR)-221-3p, pri-miR-221, GRB2-associated binding protein 1 (Gab1) expressions in hippocampal tissues were detected via RT-qPCR and Western blotting. m6A, DiGeorge syndrome critical region gene 8 (DGCR8)-bound pri-miR-221 and pri-miR-221 m6A levels were measured. The binding relationship between miR-221-3p and Gab1 was testified by dual-luciferase and RNA pull-down assays. Rescue experiments were designed to confirm the role of miR-221-3p and Gab1. METTL3 was highly expressed in CUMS rats, and silencing METTL3 attenuated cognitive deficits of CUMS rats. METTL3-mediated m6A modification facilitated processing and maturation of pri-miR-221 via DGCR8 to upregulate miR-221-3p. miR-221-3p targeted Gab1. miR-221-3p overexpression or Gab1 downregulation reversed the role of silencing METTL3 in CUMS rats. Overall, METTL3-mediated m6A modification facilitated processing and maturation of pri-miR-221 to upregulate miR-221-3p and then inhibit Gab1, thereby aggravating cognitive deficits of CUMS rats.

N6-methyladenosine modification regulates imatinib resistance of gastrointestinal stromal tumor by enhancing the expression of multidrug transporter MRP1.

N6-methyladenosine (m6A) is a frequently occurring mRNA modification, which regulates mRNA stability, splicing, and translation. However, its role in drug resistance of gastrointestinal stromal tumor (GIST) is not known. Here, we report that m6A modification levels are elevated in imatinib-resistant GIST cells and tissues, and that methyltransferase METTL3 is one of the main protein responsible for this aberrant modification. Increased METTL3 levels contributed to imatinib resistance and worse progression-free survival of GIST patients. Mechanistic studies revealed that METTL3-mediated m6A modification of the 5'UTR of the multidrug transporter MRP1 mRNA promoted drug resistance of GIST by stimulating MRP1 mRNA translation, via binding with YTHDF1 and eEF-1. Further, METTL3 transcription in imatinib resistant GIST cells was activated by ETV1, leading to the increased m6A methylation of MRP1 mRNA. This is the first report showing a novel regulatory mechanism whereby ETV1, METTL3, and the YTHDF1/eEF-1 complex mediate the translation of MRP1 mRNA in an m6A-dependent manner to regulate the intracellular concentration of imatinib and drug resistance of GIST. These findings highlight MRP1 as a new potential therapeutic target for imatinib resistance of GIST.

Maslinic acid protects against pressure-overload-induced cardiac hypertrophy by blocking METTL3-mediated m6A methylation.

Coordinated response of the heart to physiological stressors (including stress overload, ischemia, hypothyroidism, and metabolic signals) is a hallmark of heart disease. However, effective treatment and its molecular targets are unknown. Although Maslinic Acid (MA) has been shown to inhibit inflammatory responses with strong anti-tumor, anti-bacterial, and antioxidant effects, information on its role and underlying mechanism in cardiac hypertrophy are scanty. The present study revealed that 10-103 μg/ml MA treatment significantly inhibited Ang-II induced hypertrophy in NMCMs and the dosage did not influence the cell viability of H9C2 and NCMCs. Moreover, the anti-hypertrophy effect of MA (30 mg/kg·day) was verified in the TAC-induced hypertrophy mouse model in vivo. Further analysis showed that MA administration decreased the total RNA m6A methylation and METTL3 levels in Ang-II treated NMCMs and TAC stressed hearts. Rescue experiments under adenovirus-mediated myocardial METTL3 overexpression confirmed that METTL3-mediated m6A methylation is essential in M-driven inhibition of myocardial hypertrophy. Collectively, MA exerts a significant anti-hypertrophy effect by regulating the modification of METTL3-mediated m6A methylation in vitro and in vivo. These findings may provide a platform for establishing a new target and strategy for cardiac hypertrophy treatment.

Lactylation-driven METTL3-mediated RNA m6A modification promotes immunosuppression of tumor-infiltrating myeloid cells

Tumor-infiltrating myeloid cells (TIMs) are crucial cell populations involved in tumor immune escape, andtheir functions are regulated by multiple epigenetic mechanisms. The precise regulation mode of RNA N6-methyladenosine (m6A) modification in controlling TIM function is still poorly understood. Our study revealed that the increased expression of methyltransferase-like 3 (METTL3) in TIMs was correlated with the poor prognosis of colon cancer patients, and myeloid deficiency of METTL3 attenuated tumor growth in mice. METTL3 mediated m6A modification on Jak1 mRNA in TIMs, the m6A-YTHDF1 axis enhanced JAK1 protein translation efficiency and subsequent phosphorylation of STAT3. Lactate accumulated in tumor microenvironment potently induced METTL3 upregulation in TIMs via H3K18 lactylation. Interestingly, we identified two lactylation modification sites in the zinc-finger domain of METTL3, which was essential forMETTL3 to capture target RNA. Our results emphasize the importance of lactylation-driven METTL3-mediated RNA m6A modification for promoting the immunosuppressive capacity of TIMs.

METTL3 suppresses neuropathic pain via modulating N6-methyladenosine-dependent primary miR-150 processing

Methyltransferase-like 3 (METTL3)-modulated N6-methyladenosine (m6A) was recently identified as an important epigenetic regulation type during RNA processing and contributes to multiple pathological processes. Neuropathic pain (NP) is induced by a lesion of the somatosensory nervous system, and the detailed pathways by which METTL3/m6A regulated to modulate gene dysregulation and enable NP have remained unclear. Therefore, this study investigated the function of METTL3-mediated m6A methylation on miRNA maturation, and investigated how this regulation contributes to NP progression. A rat model characterized with typical NP was established by a spared nerve-injury (SNI) method. By analyzing the expression levels of METTL3 and m6A methylation, we found that METTL3, along with m6A methylation, was dramatically downregulated in NP rats in contrast to the sham ones. Functionally, enhanced METTL3 promoted the m6A methylation in total RNAs and inhibited NP progression, whereas silencing METTL3 suppressed m6A methylation and increased NP severity. Mechanistically, METTL3 accelerated miR-150 maturation via mediating m6A methylation of primiR-150 at locus 498, cooperating with the “m6A reader” YTHDF2. Meanwhile, miR-150 could directly target brain-derived neurotrophic factor (BDNF) mRNA, and the METTL3/miR-150/BDNF regulatory pathway was finally established. Clinically, we proved that serum METTL3 mRNA was also downregulated in Shingles patients with NP, suggesting its diagnostic potential. In conclusion, we demonstrated an essential function of METTL3-regulated N6-methyladenosine during NP progression via modulating primiR-150 maturation. Serum METTL3 could effectively differentiate NP patients from healthy people, and is useful for dynamic monitoring of diseases after treatment. Therefore, the METTL3/miR-150/BDNF pathway may be a promising therapeutic target for NP patients.

METTL3/IGF2BP3 axis inhibits tumor immune surveillance by upregulating N6-methyladenosine modification of PD-L1 mRNA in breast cancer

BackgroundContinual expression of PD-L1 in tumor cells is critical for tumor immune escape and host T cell exhaustion, however, knowledge on its clinical benefits through inhibition is limited in breast cancer. N6-methyladenosine (m6A) plays a crucial role in multiple biological activities. Our study aimed to investigate the regulatory role of the m6A modification in PD-L1 expression and immune surveillance in breast cancer.MethodsMeRIP-seq and epitranscriptomic microarray identified that PD-L1 is the downstream target of METTL3. MeRIP-qPCR, absolute quantification of m6A modification assay, and RIP-qPCR were used to examine the molecular mechanism underlying METTL3/m6A/IGF2BP3 signaling axis in PD-L1 expression. B-NDG and BALB/c mice were used to construct xenograft tumor models to verify the phenotypes upon METTL3 and IGF2BP3 silencing. In addition, breast cancer tissue microarray was used to analyze the correlation between PD-L1 and METTL3 or IGF2BP3 expression.ResultsWe identified that PD-L1 was a downstream target of METTL3-mediated m6A modification in breast cancer cells. METTL3 knockdown significantly abolished m6A modification and reduced stabilization of PD-L1 mRNA. Additionally, METTL3-mediated PD-L1 mRNA activation was m6A-IGF2BP3-dependent. Moreover, inhibition of METTL3 or IGF2BP3 enhanced anti-tumor immunity through PD-L1-mediated T cell activation, exhaustion, and infiltration both in vitro and in vivo. PD-L1 expression was also positively correlated with METTL3 and IGF2BP3 expression in breast cancer tissues.ConclusionOur study suggested that METTL3 could post-transcriptionally upregulate PD-L1 expression in an m6A-IGF2BP3-dependent manner to further promote stabilization of PD-L1 mRNA, which may have important implications for new and efficient therapeutic strategies in the tumor immunotherapy.

JNK Signaling Promotes Bladder Cancer Immune Escape by Regulating METTL3-Mediated m6A Modification of PD-L1 mRNA.

The identification of a novel m6A-dependent mechanism underlying immune system evasion by bladder cancer cells reveals JNK signaling as a potential target for bladder cancer immunotherapy.

N6-methyladenosine RNA modification of glutamatergic neurons is associated with contextual fear discrimination

Fear memory overgeneralization is a hallmark of many stress-related disorders, especially posttraumatic stress disorder. The neurobiology of fear memory generalization and discrimination involves a series of interplays between molecular and cellular factors, the mechanisms of which remain largely unexplored. N6-methyladenosine (m6A) of RNA is a reversible and dynamically regulated posttranscriptional modification with especially high levels in mammalian brain. In the present study, we found a positive correlation of m6A methylation abundance with accurate threat discrimination ability in response to fear memory. In addition, the methyltransferase Mettl3 levels showed a significant positive correlation with fear discrimination ability, suggesting a vital role of hippocampal METTL3-mediated m6A modification on contextual fear memory discrimination. By generating cell type-specific Mettl3 deficient mouse models, we demonstrated that METTL3 expressed in hippocampal glutamatergic neurons, but not in GABAergic neurons or astrocytes is specifically involved in fear discrimination and memory generalization, although Mettl3 depletion failed to affect the capability of developing fear memory. Taken together, our study revealed that m6A tagging is a crucial regulator of fear memory generalization through finetuning the activity of glutamatergic neurons.

TBK1-METTL3 axis facilitates antiviral immunity.

mRNA m6A modification is heavily involved in modulation of immune responses. However, its function in antiviral immunity is controversial, and how immune responses regulate m6A modification remains elusive. We here find TBK1, a key kinase of antiviral pathways, phosphorylates the core m6A methyltransferase METTL3 at serine 67. The phosphorylated METTL3 interacts with the translational complex, which is required for enhancing protein translation, thus facilitating antiviral responses. TBK1 also promotes METTL3 activation and m6A modification to stabilize IRF3 mRNA. Type I interferon (IFN) induction is severely impaired in METTL3-deficient cells. Mettl3fl/fl-lyz2-Cre mice are more susceptible to influenza A virus (IAV)-induced lethality than control mice. Consistently, Ythdf1-/- mice show higher mortality than wild-type mice due to decreased IRF3 expression and subsequently attenuated IFN production. Together, we demonstrate that innate signals activate METTL3 via TBK1, and METTL3-mediated m6A modification secures antiviral immunity by promoting mRNA stability and protein translation.

Mechanism of METTL3-Mediated m6A Modification in Cardiomyocyte Pyroptosis and Myocardial Ischemia-Reperfusion Injury.

Myocardial ischemia/reperfusion (MI/R) injury is a complicated pathophysiological process associated with cardiomyocyte pyroptosis. Methyltransferase-like protein 3 (METTL3) catalyzes the formation of N6-methyl-adenosine (m6A) and participates in various biological processes. This study probed into the mechanism of METTL3 in cardiomyocyte pyroptosis in MI/R injury. A rat model of MI/R was established. Rat cardiomyocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment for the establishment of a cell model in vitro. METTL3 expression in myocardial tissues of MI/R rats and OGD/R-treated cardiomyocytes was determined using RT-qPCR and Western blot. The pathological changes of rat myocardial tissues were observed using hematoxylin and eosin staining. The positive expression of NLRP3 in myocardial tissues or cardiomyocytes was observed through immunohistochemistry or immunofluorescence. The activity of caspase-1 was measured using the colorimetric method. The expressions of GSDMD and cleaved caspase-1, as well as the levels of IL-1β and IL-18 in rat myocardial tissues or cardiomyocytes were determined. m6A modification level was quantified. The binding relationship between pri-miR-143-3p and DGCR8 and the enrichment of m6A on pri-miR-143-3p were detected. The binding relationship between miR-143-3p and protein kinase C epsilon (PRKCE) was verified. METTL3 expression was elevated in MI/R rats and OGD/R cardiomyocytes. METTL3 silencing alleviated myocardial injury, reduced the number of NLRP3-positive cardiomyocytes, suppressed caspase-1 activity, decreased the protein levels of GSDMD-N and cleaved caspase-1, and decreased IL-1β and IL-18 levels. METTL3 increased the total m6A level in MI/R rats and injured cardiomyocytes, promoted DGCR8 binding to pri-miR-143-3p, and enhanced miR-143-3p expression. miR-143-3p suppressed PRKCE transcription, and miR-143-3p overexpression reversed the inhibitory effect of METTL3 silencing on cardiomyocyte pyroptosis. METTL3 promoted DGCR8 binding to pri-miR-143-3p through m6A modification, thus enhancing miR-143-3p expression to inhibit PRKCE transcription and further aggravating cardiomyocyte pyroptosis and MI/R injury.

β-Elemene Restrains PTEN mRNA Degradation to Restrain the Growth of Lung Cancer Cells via METTL3-Mediated N6 Methyladenosine Modification.

Lung cancer is one of the most fatal malignancies and the leading cause of cancer death worldwide. β-Elemene, a well-known anticancer drug, has drawn a great deal of attention from researchers attributed to its limited side impacts. N6-Methyladenosine (m6A) modification is the most common RNA modification and plays a vital role in the pathogenesis of multiple tumors. However, the functional link between β-elemene and the m6A modification in lung cancer development remains unexplored. In this study, we investigated whether m6A modification was responsible for the impacts of β-elemene on lung cancer. Firstly, outcomes suggested that β-elemene restrained the malignant behaviors of A549 together with H1299 cells. Thereafter, we observed that β-elemene markedly regulated METTL3, YTHDF1, and YTHDC1 among various m6A modulators. METTL3 was selected for further study because of its oncogenic function in lung cancer. RT-qRCR and western blot assays exhibited that the mRNA and protein expression levels of METTL3 were lessened by the administration of β-elemene. Mechanistically, β-elemene exerted the restrictive impacts on the cell growth of lung cancer in vivo and in vitro through targeting METTL3. More importantly, β-elemene contributed to the augmented PTEN expression via suppressing its m6A modification. To sum up, we provided strong clues that β-elemene promoted PTEN expression to retard lung cancer progression by the regulation of METTL3-mediated m6A modification.

METTL3 promotes lung adenocarcinoma tumor growth and inhibits ferroptosis by stabilizing SLC7A11 m6A modification

BackgroundN6-methyladenosine (m6A) has emerged as a significant regulator of the progress of various cancers. However, its role in lung adenocarcinoma (LUAD) remains unclear. Here, we explored the biological function and underlying mechanism of methyltransferase-like 3 (METTL3), the main catalyst of m6A, in LUAD progression.MethodsThe expression of m6A, METTL3, YTHDF1 and SLC7A11 were detected by immunochemistry or/and online datasets in LUAD patients. The effects of METTL3 on LUAD cell proliferation, apoptosis and ferroptosis were assessed through in vitro loss-and gain-of-function experiments. The in vivo effect on tumorigenesis of METTL3 was evaluated using the LUAD cell xenograft mouse model. MeRIP-seq, RNA immunoprecipitation and RNA stability assay were conducted to explore the molecular mechanism of METTL3 in LUAD.ResultsThe results showed that the m6A level, as well as the methylase METTL3 were both significantly elevated in LUAD patients and lung cancer cells. Functionally, we found that METTL3 could promote proliferation and inhibit ferroptosis in different LUAD cell models, while METTL3 knockdown suppressed LUAD growth in cell-derived xenografts. Mechanistically, solute carrier 7A11 (SLC7A11), the subunit of system Xc−, was identified as the direct target of METTL3 by mRNA-seq and MeRIP-seq. METTL3-mediated m6A modification could stabilize SLC7A11 mRNA and promote its translation, thus promoting LUAD cell proliferation and inhibiting cell ferroptosis, a novel form of programmed cell death. Additionally, we demonstrated that YTHDF1, a m6A reader, was recruited by METTL3 to enhance SLC7A11 m6A modification. Moreover, the expression of YTHDF1 and SLC7A11 were positively correlated with METTL3 and m6A in LUAD tissues.ConclusionsThese findings reinforced the oncogenic role of METTL3 in LUAD progression and revealed its underlying correlation with cancer cell ferroptosis; these findings also indicate that METTL3 is a promising novel target in LUAD diagnosis and therapy.

N(6)-methyladenosine RNA methyltransferase like 3 inhibits extracellular matrix synthesis of endplate chondrocytes by downregulating sex-determining region Y-Box transcription factor 9 expression under tension

Tension stimulation is an important inducer of endplate cartilage degeneration, but the specific regulatory mechanism remains unclear. This study was the first to reveal the mechanism by which methyltransferase-like 3 (METTL3)-mediated N(6)-methyladenosine (m6A) modification affected the extracellular matrix anabolism by tension-induced endplate chondrocytes. We examined the differences in METTL3 expression and m6A methylation levels in human endplate chondrocytes and human cartilage endplate tissues under in vitro tension. The effect on endplate cartilage degeneration was evaluated by manipulating m6A methylation mediated by METTL3 in vivo and in vitro. The effect of METTL3-mediated m6A methylation on the stability of sex-determining region Y-box transcription factor 9 (SOX9) gene expression was determined experimentally. METTL3 expression and m6A methylation levels were significantly increased in degenerative human endplate cartilage tissue. Similarly, tension stimulation inhibited the ability of human endplate chondrocytes to synthesize extracellular matrix, which was accompanied by an increase in METTL3-mediated m6A methylation. The ability of endplate chondrocytes to resist tension was significantly enhanced by inhibiting METTL3 expression and subsequently downregulating m6A methylation in vitro and in vivo, thereby reducing intervertebral disc degeneration. Furthermore, METTL3 mediated SOX9 RNA methylation and disrupted SOX9 mRNA stability, thereby inhibiting the gene expression of the downstream collagen type II alpha 1 chain. Tension stimulation downregulated SOX9 expression through METTL3-mediated m6A methylation, thereby inhibiting the synthesis of extracellular matrix in endplate chondrocytes.

METTL3-mediated m6A modification of TIMP2 mRNA promotes podocyte injury in diabetic nephropathy

Epigenetic changes are present in many physiological and pathological processes. The N6-methyladenosine (m6A) modification is the most common modification in eukaryotic mRNA. However, the role of m6A modification in diabetic nephropathy (DN) remains elusive. Here, we found that m6A modification was significantly upregulated in the kidney of type 1 and type 2 diabetic mice, which was caused by elevated levels of METTL3. Moreover, METTL3 is increased in podocyte of renal biopsy from patients with DN, which is related to renal damage. METTL3 knockout significantly reduced the inflammation and apoptosis in high glucose (HG)-stimulated podocytes, while its overexpression significantly aggravated these responses invitro. Podocyte-conditional knockout METTL3 significantly alleviated podocyte injury and albuminuria in streptozotocin (STZ)-induced diabetic mice. Therapeutically, silencing METTL3 with adeno-associated virus serotype-9 (AAV9)-shMETTL3 invivo mitigated albuminuria and histopathological injury in STZ-induced diabetic mice and db/db mice. Mechanistically, METTL3 modulated Notch signaling via the m6A modification of TIMP2 in an insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2)-dependent manner and exerted pro-inflammatory and pro-apoptotic effects. In summary, this study suggested that METTL3-mediated m6A modification is an important mechanism of podocyte injury in DN. Targeting m6A through the writer enzyme METTL3 is a potential approach for the treatment of DN.

RNA methylation-mediated LINC01559 suppresses colorectal cancer progression by regulating the miR-106b-5p/PTEN axis.

Long noncoding RNAs (lncRNAs) regulate multiple biological effects in cancers. Recently, RNA methylation has been found to modify not only coding RNAs but also some noncoding RNAs. How RNA methylation affects lncRNAs to affect colorectal cancer (CRC) progression remains elusive. The expression of LINC01559 was explored through RNA sequencing, quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). The preliminary exploration of its function was performed using Western blotting (WB) and immunohistochemistry (IHC). Functional experiments in vitro and in vivo were conducted to explore the biological functions of LINC01559 in CRC. The LINC01559/miR-106-5p/PTEN axis was verified through fluorescence in situ hybridization (FISH), luciferase assays, and rescue experiments. RIP-sequencing, m6A RNA immunoprecipitation (MeRIP) assays and bioinformatic analysis were conducted to determine the upstream mechanism of LINC01559. The results showed that LINC01559 was downregulated in CRC compared with normal controls. Lower expression of LINC01559 in CRC patients predicted a poor prognosis. In addition, PTEN was found to be positively correlated with LINC01559, and miR-106b-5p could be the link between LINC01559 and PTEN. Then, silencing LINC01559 restored the malignant phenotype of CRC cells, while cotransfection of miR-106b-5p inhibitor neutralized this effect. Mechanistically, we found abundant m6A modification sites on LINC01559. Then, we uncovered these sites as potential targets of METTL3 through experiments in vivo. The results revealed a negative functional regulation of the LINC01559/miR-106b-5p/PTEN axis in CRC progression and explored a new mechanism of METTL3-mediated m6A modification on LINC01559. These results elucidate a novel potential therapeutic target for CRC treatment.