JOURNAL/nrgr/04.03/01300535-202602000-00042/figure1/v/2025-05-05T160104Z/r/image-tiff The neuroinflammatory response mediated by microglial activation plays an important role in the secondary nerve injury of traumatic brain injury. The post-transcriptional modification of N 6 -methyladenosine is ubiquitous in the immune response of the central nervous system. The fat mass and obesity-related protein catalyzes the demethylation of N 6 -methyladenosine modifications on mRNA and is widely expressed in various tissues, participating in the regulation of multiple diseases' biological processes. However, the role of fat mass and obesity in microglial activation and the subsequent neuroinflammatory response after traumatic brain injury is unclear. In this study, we found that the expression of fat mass and obesity was significantly down-regulated in both lipopolysaccharide-treated BV2 cells and a traumatic brain injury mouse model. After fat mass and obesity interference, BV2 cells exhibited a pro-inflammatory phenotype as shown by the increased proportion of CD11b + /CD86 + cells and the secretion of pro-inflammatory cytokines. Fat mass and obesity-mediated N 6 -methyladenosine demethylation accelerated the degradation of ADAM17 mRNA, while silencing of fat mass and obesity enhanced the stability of ADAM17 mRNA. Therefore, down-regulation of fat mass and obesity expression leads to the abnormally high expression of ADAM17 in microglia. These results indicate that the activation of microglia and neuroinflammatory response regulated by fat mass and obesity-related N 6 -methyladenosine modification plays an important role in the pro-inflammatory process of secondary injury following traumatic brain injury.

Although N6-methyladenosine (m6A) may be related to the pathogenesis of fibrotic process, the mechanism of m6A modification in aging-related idiopathic pulmonary fibrosis (IPF) remains unclear. Three-milliliter venous blood was collected from IPF patients and healthy controls. MeRIP-seq and RNA-seq were utilized to investigate differential m6A modification. The expressions of identified m6A regulator and target gene were validated using MeRIP-qPCR and real-time PCR. Moreover, we established an animal model and a senescent model of A549 cells to explore the associated molecular mechanism. Our study provided a panorama of m6A methylation in IPF. Increased peaks (3756) and decreased peaks (4712) were observed in the IPF group. The association analysis showed that 749 DEGs were affected by m6A methylation in IPF. Among the m6A regulators, the expression of METTL14 decreased in IPF. The m6A level of our interested gene DDIT4 decreased significantly, but the mRNA level of DDIT4 was higher in IPF. This was further verified in bleomycin-induced pulmonary fibrosis. At the cellular level, it was further confirmed that METTL14 and DDIT4 might participate in the senescence of alveolar epithelial cells. The downregulation of METTL14 might inhibit the decay of DDIT4 mRNA by reducing the m6A modification level of DDIT4 mRNA, leading to high expression of DDIT4 mRNA and protein. Our study provided a panorama of m6A alterations in IPF and discovered METTL14 as a potential intervention target for epigenetic modification in IPF. These results pave the way for future investigations regarding m6A modifications in aging-related IPF.

The crosstalk between the tumour immune microenvironment (TIME) and tumour cells promote immune evasion and resistance to immunotherapy in gastrointestinal (GI) tumours. Post-transcriptional regulation of genes is pivotal to GI tumours progression, and RNA-binding proteins (RBPs) serve as key regulators via their RNA-binding domains. RBPs may exhibit either anti-tumour or pro-tumour functions by influencing the TIME through the modulation of mRNAs and non-coding RNAs expression, as well as post-transcriptional modifications, primarily N6-methyladenosine (m6A). Aberrant regulation of RBPs, such as HuR and YBX1, typically enhances tumour immune escape and impacts prognosis of GI tumour patients. Further, while targeting RBPs offers a promising strategy for improving immunotherapy in GI cancers, the mechanisms by which RBPs regulate the TIME in these tumours remain poorly understood, and the therapeutic application is still in its early stages. This review summarizes current advances in exploring the roles of RBPs in regulating genes expression and their effect on the TIME of GI tumours, then providing theoretical insights for RBP-targeted cancer therapies.

Graves' ophthalmopathy (GO) obvious manifestation is the imbalance of Th17/Treg. N6-methyladenosine (m6A) methylation is an important regulator of Th17/Treg balance. However, few reports narrate how m6A regulators mediate the role of genes in GO progression. We explored the m6A modification of THBS1 mediated by WTAP, and the mechanism by which THBS1 regulated glycolysis and Th17/Treg balance. A total of 12 peripheral blood (4 GO samples, 4 GH samples, and 4 health samples) were collected to measure the percentage of Th17/Treg in monocytes by flow cytometry. RNA sequencing (RNA-seq) combined with MeRIP sequencing (MeRIP-seq) was used to screen differentially expressed and methylated genes. MeRIP-qPCR was performed to evaluate the m6A abundance of THBS1 after WTAP silencing. Glycolysis of CD4+ T cells was reflected by the lactate content and glucose uptake. The number of Th17 cells was increased in GO peripheral blood, whereas the Treg cells decreased. RNA-seq acquired 679 differentially expressed genes (308 up-regulated, and 371 down-regulated) in the CD4+ T cells of GO compared to healthy control. MeRIP-seq identified 3277 m6A peaks between the GO group and the healthy control group, corresponding with 2744 genes (1143 hypermethylated and 1601 hypomethylated). Combined analysis of RNA-seq and MeRIP-seq showed 81 hypermethylated and up-regulated genes. Among the six candidate genes in the PI3K-signaling pathway, THBS1 was the most significantly differentially expressed and hypermethylated. THBS1 silencing resulted in decreased lactate content and glucose uptake in CD4+ T cells. WTAP was significantly upregulated in CD4+ T cells of GO, and WTAP silencing significantly reduced m6A abundance and expression of THBS1. Upregulated and hypermethylated THBS1 mediated by WTAP promoted glycolysis of CD4+ T cells, affected Th17/Treg balance, and facilitated GO progression. We provided a novel potential target for GO treatment and revealed the molecular mechanism of WTAP and THBS1 in GO under the m6A perspective.

The gut microbiota undergoes continuous variations among individuals and across their lifespan, shaped by diverse factors encompassing diet, age, lifestyle choices, medication intake, and disease states. These microbial inhabitants play a pivotal role in orchestrating physiological metabolic pathways through the production of metabolites like bile acids, choline, short-chain fatty acids, and neurotransmitters, thereby establishing a dynamic "gut-organ axis" with the host. The intricate interplay between the gut microbiota and the host is indispensable for gut health, and RNA N6-methyladenosine modification, a pivotal epigenetic mark on RNA, emerges as a key player in this process. M6A modification, the most prevalent internal modification of eukaryotic RNA, has garnered significant attention in the realm of RNA epigenetics. Recent findings underscore its potential to influence gut microbiota diversity and intestinal barrier function by modulating host gene expression patterns. Conversely, the gut microbiota, through its impact on the epigenetic landscape of host cells, may indirectly regulate the recruitment and activity of RNA m6A-modifying enzymes. This review endeavors to delve into the biological functions of m6A modification and its consequences on intestinal injury and disease pathogenesis, elucidating the partial possible mechanisms by which the gut microbiota and its metabolites maintain host intestinal health and homeostasis. Furthermore, it also explores the intricate crosstalk between them in intestinal injury, offering a novel perspective that deepens our understanding of the mechanisms underlying intestinal diseases.

Glioblastoma (GBM) was the most common and deadliest malignant brain tumor in adults, with a poor prognosis. Effective targeted drugs are still lacking, and the presence of glioblastoma stem cells (GSC) is a major factor contributing to radiotherapy resistance. Screening for targeted drugs that can sensitize GBM to radiotherapy is crucial. FTO is considered an attractive potential target for tumor therapy, as it mediates m6A demethylation to regulate the stability of target genes. In this study, we evaluated the role of FTO inhibition in promoting the sensitivity of GSC cells to radiotherapy through tumor sphere formation assays, cell apoptosis assays, and in situ GSC tumor models. We preliminarily explored the molecular mechanisms by transcriptome sequencing and m6A methylation sequencing to investigate how inhibiting FTO increases radiotherapy sensitivity. The results showed that downregulation of FTO expression or FTO inhibitor FB23-2 combined with radiotherapy significantly inhibited GSC cell proliferation and self-renewal and increased apoptosis. FB23-2 combined with radiotherapy effectively inhibited intracranial tumor growth in mice and prolonged the survival of tumor-bearing mice. Furthermore, FTO inhibition sustained the increase of γH2AX expression induced by radiotherapy while decreasing Rad51 expression. Importantly, we found that inhibiting FTO could increase m6A methylation modification of VEGFA, thereby downregulating both mRNA and protein expression of VEGFA. Our findings provide a new therapeutic strategy for enhancing GBM radiotherapy sensitivity and lay the theoretical and experimental groundwork for clinical trials targeting FTO.

RNA epigenetic modifications are crucial in tumor development, with N6-methyladenosine (m6A) being the most prevalent epigenetic modification found in all eukaryotic messenger RNAs. Accumulating evidence indicates that m6A modifications significantly influence the progression of various malignancies, including head and neck cancer (HNC). The Methyltransferase-like (METTL) family proteins, a group of methyltransferases identified in recent years, function as the "writers" of m6A modifications. These proteins affect RNA stability, translation efficiency, splicing, and localization, thereby regulating diverse cellular functions and promoting tumorigenesis in multiple cancers through their methylation domains. This review aims to summarize existing literature on the METTL family of m6A-modified RNA to elucidate their roles in HNC, providing a theoretical foundation for their potential use as therapeutic targets.

Pancreatic ductal adenocarcinoma (PDAC) is recognized globally as one of the most lethal tumours, and effective biomarkers to diagnose PDAC early are needed. ORC6, a subunit of the origin recognition complex (ORC), initiates DNA replication and ensures genomic stability. Previous studies have indicated that ORC6 is procarcinogenic in various cancers, yet its role in PDAC remains uninvestigated.

We evaluated the relationships between ORC6 expression and the clinical features of patients with PDAC with the TCGA, GTEx, and GEO databases. The role of ORC6 in PDAC cells was explored by RNA interference in vitro and in vivo. Next, we verified the effect of the METTL3/IGF2BP3/ORC6 axis on PDAC progression by western blotting, RT-qPCR, RNA immunoprecipitation, and methylated RNA immunoprecipitation. Finally, transcriptome analysis was performed to explore the influence of ORC6 on p53 in PDAC cells.

Elevated ORC6 levels were observed in PDAC cells, which correlated with poorer clinical outcomes. Both in vivo and in vitro experiments demonstrated that ORC6 knockdown suppressed proliferation and promoted apoptosis. Additionally, we demonstrated that METTL3/IGF2BP3 interacted with ORC6 mRNA via N6-methyladenosine modification to improve ORC6 mRNA stability. Transcriptomic analysis and experiments indicated that ORC6 promoted PDAC progression by inhibiting serine-15 phosphorylation in p53.

Our findings validate the role of ORC6 in PDAC and support the hypothesis that the METTL3/IGF2BP3/ORC6/p53 axis may be a novel therapeutic target for PDAC, and inhibiting this axis may be an advantageous therapeutic strategy for curing PDAC.

Abdominal aortic aneurysm (AAA) is a type of cardiovascular disease. Sudden aortic rupture and subsequent bleeding are the main causes of mortality due to AAA. N6‑methyladenosine (m6A) methylation, the most common epitranscriptomic modification in eukaryotic mRNAs, has a key role in the regulation of gene expression. m6A methylation markedly influences the development and progression of AAA. The present review highlights the mechanism of m6A methylation in AAA, including current research progress and future prospects. From a mechanistic perspective, m6A methylation exerts its influence on AAA‑related genes by modulating the post‑transcriptional levels of RNA, thereby impacting the pathological process of AAA. In terms of clinical applications, the mechanisms by which m6A methylation regulators influence their development and progression in AAA involve multiple target genes and signaling pathways. These regulatory factors affect inflammatory immunomodulation, cell proliferation, apoptosis and endogenous processes by modulating the m6A modification status of target genes and the activity of immune‑related signaling pathways. Therefore, for the prevention and treatment of AAA, current therapeutic strategies should comprehensively consider the interactions and synergistic regulation among m6A methylation regulators to reveal the integrated effects of the entire regulatory network in AAA development. Consequently, a more comprehensive understanding of the precise mechanisms of m6A methylation in AAA should be attained, which will support the development of innovative therapeutic strategies aimed at m6A methylation and establish a basis for the early diagnosis and treatment of AAA.

Acute kidney injury (AKI) lacks a definitive therapeutic approach beyond supportive care. One significant pathological mechanism involves the regulated death of tubular epithelial cells; however, the regulatory mechanisms underlying this cell death pathway require further investigation. The N6-methyladenosine (m6A) modification, recognized as the most prevalent modification in eukaryotes, plays a critical role in the regulatory processes associated with AKI. Here, this study investigates the association between methyltransferase-like 3 (METTL3) and pyroptosis in mice with folic acid (FA)-induced AKI. Both in vitro and in vivo experiments have confirmed that METTL3 plays a role in AKI progression, correlating with renal epithelial cell pyroptosis and inflammation. Moreover, RNA immunoprecipitation quantitative PCR (RIP-qPCR) analysis demonstrated that METTL3-mediated m6A methylation occurred in the mRNA of Apoptosis-associated speck-like protein containing a CARD (ASC) in H2O2-induced renal tubular epithelial (TCMK-1) cells. Notably, METTL3 knockdown resulted in reduced ASC protein expression, decreased release of inflammatory factors, and reduced pyroptosis. In addition, we verified the inhibitory effect of berberine hydrochloride, a monomer used in traditional Chinese medicine, on METTL3 expression. We also demonstrated that berberine ameliorated FA-induced AKI and H2O2-induced pyroptosis in TCMK-1 cells by inhibiting METTL3 and modulating the ASC/caspase-1/Gasdermin D axis. These findings provide insights into targeted therapies and drug development for AKI.

Liver cancer refers to malignant tumors that form in the liver and is usually divided into several types, the most common of which is hepatocellular carcinoma (HCC), which originates in liver cells. Other rare types of liver cancer include intrahepatic cholangiocarcinoma (iCCA). m6A modification is a chemical modification of RNA that usually manifests as the addition of a methyl group to adenine in the RNA molecule to form N6-methyladenosine. This modification exerts a critical role in various biological processes by regulating the metabolism of RNA, affecting gene expression. Recent studies have shown that m6A modification is closely related to the occurrence and development of liver cancer, and m6A regulators can further participate in the pathogenesis of liver cancer by regulating the expression of key genes and the function of specific cells. In this review, we provided an overview of the latest advances in m6A modification in liver cancer research and explored in detail the specific functions of different m6A regulators. Meanwhile, we deeply analyzed the mechanisms and roles of m6A modification in liver cancer, aiming to provide novel insights and references for the search for potential therapeutic targets. Finally, we discussed the prospects and challenges of targeting m6A regulators in liver cancer therapy.

The RNA methyltransferase methyltransferaselike protein 16 (METTL16) is upregulated in a large proportion of hepatocellular carcinoma (HCC), and its high expression is associated with poor clinical outcomes. METTL16 deletion inhibits HCC growth in vitro and in vivo. Referencing the structure of cinnamic acid, here we designed and synthesized a novel series of small molecular compounds, and found through bioactivity screening that compound 15a effectively reduced METTL16 level and modulated oncogenic PI3K/AKT pathway signaling. Compound 15a inhibited the proliferation and migration of HepG2 cells, and induced apoptosis in vitro. Furthermore, compound 15a significantly inhibited the growth of patient-derived HCC xenografts in nude mice with greater efficacy than the multi-kinase inhibitor lenvatinib. The promising efficacy and good biosafety profile of compound 15a enables us to further develop this compound for treating patients with HCC and possibly other cancers in clinic.

N6-methyladenosine (m6A) methylation is the most prevalent RNA modification that is regulated by three regulatory factors: "writers", "erasers" and "readers". m6A modification regulates RNA stability and other mechanisms, including translation, cleavage, and degradation. Current research has demonstrated that m6A methylation is involved in the regulation of occurrence and development of cancers by controlling the expression of cancer-related genes. This review summarizes the role of m6A modification on messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) in cervical cancer (CC). We highlight the dual role of m6A regulatory factors, which act as oncogenes or tumor suppressors depending on the cellular context and downstream targets. Additionally, we examine how ncRNAs reciprocally regulate m6A modification in two ways: by guiding the deposition or removal of m6A modifications on RNA targets, and by modulating the expression of m6A regulatory factors. These interactions further contribute to tumor progression. Furthermore, the therapeutic potential of targeting m6A modification has been emphasized in CC. Moreover, recent advances in small-molecule inhibitors targeting m6A regulators and RNA-based therapies which may offer new treatment strategies have been summarized. Finally, we discuss the current challenges in m6A modification research and provide suggestions for future research directions. This review aims to deepen the understanding of m6A modification in CC and contribute to the development of targeted and personalized treatment strategies.

Gastrointestinal (GI) cancers remain a leading cause of cancer-related mortality worldwide, with metabolic reprogramming recognized as a central driver of tumor progression and therapeutic resistance. Among the key regulatory layers, N6-methyladenosine (m6A) RNA modification-mediated by methyltransferases (writers such as METTL3/14), RNA-binding proteins (readers like YTHDFs and IGF2BPs), and demethylases (erasers including FTO and ALKBH5), plays a pivotal role in controlling gene expression and metabolic flux in the tumor context. Concurrently, the gut microbiota profoundly influences GI tumorigenesis and immune evasion by modulating metabolite availability and remodeling the tumor microenvironment. Recent evidence has uncovered a bidirectional crosstalk between microbial metabolites and m6A methylation: microbiota-derived signals dynamically regulate m6A deposition on metabolic and immune transcripts, while m6A modifications, in turn, regulate the stability and translation of key mRNAs such as PD-L1 and FOXP3. This reciprocal interaction forms self-reinforcing epigenetic circuits that drive tumor plasticity, immune escape, and metabolic adaptation. In this review, we dissect the molecular underpinnings of the microbiota-m6A-metabolism axis in GI cancers and explore its potential to inform novel strategies in immunotherapy, metabolic intervention, and microbiome-guided precision oncology.

Antigen-specific effector CD4+ T cells are critical for defense against exogenous pathogens. However, the epigenetic mechanisms underlying CD4+ T cell immune responses, particularly RNA modifications, remain incompletely understood. In this study, we employed a T cell-specific deletion of the fat mass and obesity-associated protein (FTO), a key N6-methyladenosine (m6A) demethylase, to elucidate its role in CD4+ T cell mediated immunity. Our findings demonstrate that FTO is essential for maintaining CD4+ T cell immune responses and protective functions. Specifically, FTO deficiency restricts the expansion of CD4+ T helper (Th)1 effector cells following antigen challenge and results in decreased expression of T-bet and IFN-γ in Th1 cells. Additionally, FTO deficient CD4+ T cells exhibit impaired pathogen elimination. Collectively, our study reveals a novel epigenetic regulatory mechanism in supporting CD4+ T cell differentiation, providing new insights into the post-transcriptional regulation of CD4+ T cell immunity and highlighting the potential for therapeutic strategies.

Human cytomegalovirus (HCMV) is a prevalent pathogen that chronically infects the majority of human population. Among the many features that allow such widespread HCMV infection, one is its ability to maintain a transcriptionally dormant immune-evasive state called latency by suppressing its own major immediate early promoter (MIEP) via epigenetic alterations. In this study, we show a mechanism of MIEP regulation in which the major immediate early (MIE) gene product, immediate early 1 (IE1) transcript, downregulates its own promoter activity in an m6A modification-dependent manner. We found that the loss of the m6A writer, METTL3, in host cells impedes latency establishment in these cells. Through transcriptome-wide m6A profiling of latently infected monocytes, we identified that the major immediate early gene product IE1 transcript is m6A-modified during latent infection. Using IE1-specific m6A-abolished mutants, we found that m6A modification of the IE1 transcript was necessary for the efficient repression of MIEP, and these mutant viruses exhibited a significant defect in establishing latency and progressed toward lytic-like infection in the human monocytic cell line (THP-1) and primary CD14+ monocytes. Our findings demonstrate that HCMV exploits the host m6A machinery to suppress its own lytic program to establish latency and uncover an unexpected role of immediate early gene messenger RNA (mRNA) in regulating its own expression.

N6-Methyladenosine (m6A) ranks among the most prevalent modifications in RNA, which serves as a biomarker for diseases, such as lung cancer. Herein, we developed a CRISPR/Cas13a-Csm6 tandem assay (termed CRISPRm6A assay) allowing for preamplification-free, sensitive, and rapid detection of RNA m6A modifications. The coupling of Cas13a-Csm6 tandem with MazF endoribonuclease enables the assay to identify m6A RNA with single-base resolution. Compared to the CRISPRm6A assay using Cas13a alone, the tandem CRISPRm6A assay yielded an improved sensitivity for RNA detection by ∼22 times, thus enabling preamplification-free detection of RNA m6A. Particularly, the proposed assay enabled quantification of m6A abundance down to 0.5% at the picomole level in lncRNA MALAT1 and demonstrated a 100% correlation in diagnosing nonsmall cell lung cancer. In summary, the CRISPRm6A assay supports two key applications in biological samples: (1) precise determination of m6A sites and (2) quantitative measurement of m6A fractions. Therefore, the CRISPR tandem method presents a promising tool for RNA epigenetics-based diagnostics.

Diabetic kidney disease (DKD) is a common complication of diabetes. N6-Methyladenosine (m6A) modification is a widely studied epigenetic mechanism. Methyltransferase-like (METTL) 3 is a well-studied methyltransferase. This study aimed to investigate the role of METTL3 in DKD and the underlying mechanism.

Thirty-five DKD patients and 28 control volunteers were recruited. Animal and cell DKD models were established. QRT-PCR and Western blot were performed to analyze the expression of METTL3 and fibrosis-related indicators. Cell viability and proliferation were assessed via a cell counting kit-8 and colony formation assays. Ferrous iron (Fe2+), malonaldehyde (MDA), and glutathione (GSH) contents were measured by commercial kits. The interaction between METTL3/YTH N6-methyladenosine RNA binding protein (YTHDF)3 and transferrin receptor-1 (TfR1) was examined through RNA immunoprecipitation and dual-luciferase reporter assays.

Results showed that METTL3-mediated m6A modification was elevated in kidney tissues of DKD patients and in high glucose (HG)-treated human renal mesangial cells (HRMCs). Besides, HG-treated HRMCs showed increased ferroptosis. In addition, METTL3 inhibition increased cell proliferation and inhibited ferroptosis in HRMCs. Mechanically, the METTL3/YTHDF3 m6A axis enhanced the stability of TfR1 mRNA. Moreover, YTHDF3 inhibition increased cell proliferation and inhibited ferroptosis in HRMCs. Finally, in vivo study results indicated that METTL3 deficiency inhibited ferroptosis and improved pathological damages.

In summary, METTL3/YTHDF3 m6A axis promoted ferroptosis in DKD by stabilizing TfR1, which could provide a reference for DKD treatment.

This work reported the neuronal protection of low-dose lipopolysaccharide (LD-LPS) after spinal cord injury (SCI). SCI rat model was constructed, after adenovirus-mediated ALKBH5 vectors and shRNA transfection and LD-LPS pre-treatment. Hematoxylin and eosin, Nissl, TUNEL staining of spinal cord tissues were adopted to monitor pathological changes, neuronal survival and apoptosis. PC12 cells transfected with ALKBH5 vectors and ALKBH5/Nrf2 siRNAs were treated by LD-LPS, followed by oxygen and glucose deprivation/reoxygenation (OGD/R). Cell viability and apoptosis were assessed by cell counting kit-8 and TUNEL assays. Neuronal oxidative stress was evaluated by appraising MDA and SOD levels. ALKBH5 and Nrf2 expression was monitored through immunohistochemistry, Western blot and qRT-PCR. Methylated RNA immunoprecipitation assay and Dot-blot experiment were for Nrf2 m6A modification detection, while RNA pull-down assay was for the binding validation between ALKBH5 and Nrf2. In rats with SCI, LD-LPS relieved spinal cord tissue damage and neuronal apoptosis; enhanced neuronal survival; decreased MDA content; elevated SOD activity; down-regulated ALKBH5; up-regulated Nrf2; and facilitated Nrf2 m6A methylation. These above influences by LD-LPS were eliminated by ALKBH5. Similar results were found in the OGD/R-induced PC12 cells after LD-LPS treatment. ALKBH5 significantly blocked Nrf2 m6A methylation, and pulled down Nrf2 protein. In the OGD/R-induced PC12 cells, the repressed oxidative stress and apoptosis by ALKBH5 silencing was abrogated by Nrf2 knockdown. LD-LPS might alleviate neuronal apoptosis and oxidative stress after SCI by facilitating Nrf2 m6A methylation via reducing ALKBH5. It was proposed to be a novel strategy for SCI treatment.

N6-methyladenosine (m6A) modification, mediated by its associated regulatory proteins, has been increasingly recognized for its involvement in diverse pathological conditions. The rostral ventrolateral medulla (RVLM), a key vasomotor center, plays a crucial role in regulating hypertension. However, alterations in m6A and associated regulators including YTHDF3 within the RVLM, along with their functional contributions to hypertension development, remain to be fully elucidated. Here, we identified that YTHDF3 levels were significantly higher in the RVLM of SHRs than in WKY rats. YTHDF3 knockdown in the RVLM of SHRs reduced neuronal excitability, sympathetic tone, and blood pressure (BP). Mechanistically, YTHDF3 promoted the degradation of XRCC1 mRNA in an m6A-dependent manner. YTHDF3 silencing increased XRCC1 expression, facilitating the repair of neuronal DNA oxidative damage and suppressing neuronal apoptosis in vitro and in vivo. These beneficial effects were abrogated by XRCC1 inhibition. Notably, XRCC1 downregulation significantly reversed the suppressive effects on RVLM neuronal excitability, sympathetic tone, and BP in SHRs caused by YTHDF3 repression. The study established, for the first time, the significance of YTHDF3 as a key regulatory factor in the neural regulation of hypertension. Targeting the YTHDF3-XRCC1 axis in the RVLM represents a promising therapeutic strategy for hypertension.

RNA modifications are crucial for post-transcriptional gene regulation. Research on RNA modifications has become a novel frontier of epitranscriptomics. Up to now, over 170 kinds of modifications have been identified on mRNA and diverse non-coding RNA. Three classes of proteins (writers, erasers, and readers) regulate the addition, removal, and identification of epigenetic marks, thus affecting RNA biological functions. Increasing evidence identifies the dysregulation of RNA modifications in different cancer types and the therapeutic potential of targeting RNA-modifying enzymes. The ability of RNA modifications to improve mRNA stability and translation efficacy and decrease immunogenicity has been exploited for the clinical use of mRNA cancer vaccines. This review aims to shed light on several vital cap, tail, and internal modifications of RNA with a focus on the connection between RNA epigenetic pathways and cancer pathogenesis. We further explore the clinical potential of RNA modifications as dynamic biomarkers for cancer diagnosis, prognosis, and therapeutic response prediction, addressing both technological challenges and translational opportunities. Finally, we analyze the limitations of current studies and discuss the research focus in the future.

N6-methyladenosine (m6A), the most abundant RNA modification in eukaryotic cells, exerts a critical influence on RNA function and gene expression. It has attracted considerable attention within the rapidly evolving field of epitranscriptomics. METTL3 is a key enzyme for m6A modification and is essential for maintaining normal m6A levels. High expression of METTL3 is closely associated with various cancers, including gastric cancer, liver cancer, and leukemia. Inhibiting METTL3 has shown potential in slowing cancer progression, thereby driving the development of METTL3 inhibitors. In this work, we summarize recent advancements in the development of METTL3 inhibitor, with a focus on medicinal chemistry strategies employed during discovery and optimization phases. We explore the application of structure-activity relationship (SAR) studies and protein-targeted degradation techniques, while addressing key challenges associated with their characterization and clinical translation. This review underscores the therapeutic potential of METTL3 inhibitors in modulating epitranscriptomic pathways and aims to offer perspectives for future research in this rapidly evolving field.

The pregnane X receptor (PXR) activator rifampicin (RIF) plays a critical role in drug-drug interactions (DDIs) by inducing cytochrome P450 (CYP) 3A4 expression. Increasing evidence indicates that n6-methyladenosine (m6A) modification is involved in the regulation of CYP basal expression. Here, we showed its effect on RIF-induced CYP3A4 expression. This study found that m6A levels and methyltransferase-like 3 (METTL3) expression were upregulated in HepG2 and LS174T cells treated with RIF, as well as in mice treated with pregnenolone-16α-carbonitrile (PCN, a typical PXR activator in mice), associated with the expression of CYP3A4 and Cyp3a11 (mouse homolog of human CYP3A4). Specifically, knockdown or overexpression of METTL3 downregulated and upregulated the basal and RIF-induced expression of CYP3A4, respectively. Similar results were obtained in a mouse model with liver-specific knockdown of Mettl3 (homolog of human METTL3) treated with PCN, indicating the involvement of m6A methylation in RIF-induced CYP3A4 expression. Mechanistically, METTL3 promotes PXR nuclear translocation and protein translation, while potentially affecting CYP3A4 mRNA stability through its binding to CYP3A4 mRNA (enhanced by RIF), thereby contributing to RIF-induced upregulation of CYP3A4 expression. Functionally, we observed that METTL3 knockdown or treatment with the METTL3 inhibitor STM2457 reduced the cytotoxicity induced by RIF combined with ritonavir. In conclusion, this study identified a novel mechanism of m6A modification in the regulation of RIF-induced CYP3A4 expression, providing valuable insights into CYP3A4-mediated DDIs.

N6-methyladenosine (m6A) methylation is the most prevalent and abundant internal modification of mRNAs and is catalyzed by the methyltransferase complex. Methyltransferase-like 3 (METTL3), the best-known m6A methyltransferase, has been confirmed to function as a multifunctional regulator in the reversible epitranscriptome modulation of m6A modification according to follow-up studies. Accumulating evidence in recent years has shown that METTL3 can regulate a variety of functional genes, that aberrant expression of METTL3 is usually associated with many pathological conditions, and that its expression regulatory mechanism is related mainly to its methyltransferase activity or mRNA posttranslational modification. In this review, we discuss the regulatory functions of METTL3 in various diseases, including metabolic diseases, cardiovascular diseases, and cancer. We focus mainly on recent progress in identifying the downstream target genes of METTL3 and its underlying molecular mechanisms and regulators in the above systems. Studies have revealed that the use of METTL3 as a therapeutic target and a new diagnostic biomarker has broad prospects. We hope that this review can serve as a reference for further studies.

Chronic diabetic wounds are a prevalent and severe complication of diabetes, contributing to higher rates of limb amputations and mortality. N6-methyladenosine (m6A) is a common RNA modification that has been shown to regulate tissue repair and regeneration. However, whether targeting m6A could effectively improve chronic diabetic wound healing remains largely unknown. Here, we found a significant reduction in mRNA m6A methylation levels within human diabetic foot ulcers, and the expression level of fat mass and obesity-associated protein (FTO) was significantly increased. We identified that m6A modifies the RNA of matrix Metalloproteinase 9 (MMP9), a key factor in diabetic wound healing, to regulate its expression. Importantly, we developed a ROS-scavenging nanocolloidal hydrogel loaded with an FTO inhibitor to increase the m6A level of MMP9 RNA in wounds. The hydrogel can effectively accelerate wound healing and skin appendage regeneration in streptozotocin-induced type I diabetic rats at day 14 (approximately 98 % compared to 76.98 % in the control group) and type II diabetic db/db mice at day 20 (approximately 93 % compared to 60 % in the control group). Overall, our findings indicate that targeting m6A with ROS-scavenging hydrogel loaded with FTO inhibitor may be an effective therapeutic strategy for diabetic wound healing.

Vascular calcification (VC) is a major adverse cardiovascular event in chronic kidney disease (CKD) patients. N6-methyladenosine (m6A) modification is vital for many biological processes, but its function and possible molecular mechanisms in VC are poorly understood. This study aimed to clarify the function and molecular mechanisms of N6-adenosine-methyltransferase-like 3 (METTL3) in VC.

The results of the bioinformatic analysis showed that METTL3 expression was significantly upregulated in calcified VSMCs. This finding was corroborated by phosphate-induced VSMCs calcification models and 5/6 nephrectomy-induced CKD mouse VC models. Afterward, Alizarin Red S staining and m6A dot blot analysis demonstrated METTL3 overexpression elevated m6A levels and encouraged calcification in VSMCs and mouse aortic rings, while METTL3 knockdown decreased m6A levels and inhibited calcium deposition in these experimental models. Furthermore, METTL3 promoted VC via the PTEN/AKT pathway, and MeRIP verified that METTL3 induced PTEN mRNA degradation by modifying it with m6A. In addition, molecular docking simulations and DARTS assays revealed that quercetin is a natural small-molecule inhibitor of METTL3. The current investigation demonstrated that quercetin mitigated VC by reducing METTL3-dependent m6A levels in vivo and in vitro.

In conclusion, this study unraveled the pathogenic mechanism of METTL3-mediated m6A modification in VC and provided new insights to establish METTL3 as a therapeutic target for VC.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy associated with poor prognosis. Baicalein, a flavonoid extracted from the roots of Scutellaria baicalensis, traditionally used in Chinese medicine, has demonstrated potential in inhibiting cancer development and progression. However, its mechanism of action remains poorly understood, particularly regarding epigenetic gene regulation through m6A RNA methylation. In this study, three human PDAC cell lines and one nonmalignant cell line were employed. The effects of baicalein were examined using multiple assays, including RT-qPCR, MeRIP-qPCR, Western blotting, spheroid formation, RNA stability, and MTT, to evaluate cellular functions and m6A regulation. Baicalein significantly reduced cell viability, migration, invasion, and colony formation. It also downregulated FTO, an enzyme critical for m6A RNA demethylation. Knockdown of FTO replicated the effects of baicalein, underscoring its oncogenic role in PDAC. Bioinformatic analysis identified ZEB1-a key transcription factor in epithelial-to-mesenchymal transition-as an m6A-modified target regulated by FTO. Both baicalein treatment and FTO knockdown enhanced m6A modification and decreased ZEB1 mRNA stability, thereby suppressing stemness-related features. Rescue experiments further confirmed that baicalein disrupts the TGF-β/FTO/ZEB1 signaling axis, highlighting its therapeutic potential in PDAC. This study offers fundamental insights for the development of novel therapeutic strategies targeting PDAC.

Long non-coding RNAs (lncRNAs) are key regulators of gene expression at transcriptional and post-transcriptional levels and serve roles in tumour progression, cancer diagnosis and prognosis. Among these, family with sequence similarity 83 member H-antisense RNA 1 (FAM83H-AS1) is an oncogenic lncRNA with elevated expression in several malignancies. FAM83H-AS1 promotes cancer cell proliferation, inhibits apoptosis, enhances migration and contributes to chemoresistance through interactions with microRNA (miR)-136-5p, miR-545-3p, miR-15a miR-10a-5p and signalling pathways such as Wnt/β-catenin and Notch receptor. FAM83H-AS1 may be a promising biomarker for cancer diagnosis and prognosis. The present review summarises the expression, mechanism and potential clinical application of FAM83H-AS1 in cancer diagnosis and prognosis.

CSRP1 (Cysteine and Glycine-Rich Protein 1) is a protein often overactivated in various cancers, promoting cell proliferation and survival, making it a key factor in cancer development. However, it is worth noting that the effect of this protein on the glycolysis process in Acute Myeloid Leukemia (AML) has not yet been studied. This study aims to investigate the role of the METTL3/YTHDF1 axis in regulating Glycolysis and its impact on AML progression by stabilizing CSRP1 mRNA. We analyzed CSRP1 expression in AML tissues and cell lines using quantitative real-time PCR (qRT-PCR) and Western blotting. Functional assays, including cell viability, colony formation, glycolysis related indicators, were performed to assess the impact of CSRP1 knockdown or overexpression on AML cells. RNA immunoprecipitation (RIP) and RNA stability assays were conducted to elucidate the mechanism of METTL3/YTHDF1-mediated regulation of CSRP1 mRNA. CSRP1 was significantly upregulated in AML tissues and cell lines. Knockdown of CSRP1 inhibited AML cell proliferation and glycolysis. Overexpression of CSRP1 promoted AML cell survival. Mechanistically, METTL3 enhanced CSRP1 mRNA stability via m6A modification, recognized and bound by YTHDF1, preventing mRNA degradation. The METTL3/YTHDF1/ CSRP1 axis plays a critical role in AML progression by regulating glycolysis. Targeting this pathway may provide a novel therapeutic strategy for AML treatment.

JOURNAL/nrgr/04.03/01300535-202506000-00026/figure1/v/2024-08-05T133530Z/r/image-tiff Spinal cord injury typically causes corticospinal tract disruption. Although the disrupted corticospinal tract can self-regenerate to a certain degree, the underlying mechanism of this process is still unclear. N6-methyladenosine (m6A) modifications are the most common form of epigenetic regulation at the RNA level and play an essential role in biological processes. However, whether m6A modifications participate in corticospinal tract regeneration after spinal cord injury remains unknown. We found that expression of methyltransferase 14 protein (METTL14) in the locomotor cortex was high after spinal cord injury and accompanied by elevated m6A levels. Knockdown of Mettl14 in the locomotor cortex was not favorable for corticospinal tract regeneration and neurological recovery after spinal cord injury. Through bioinformatics analysis and methylated RNA immunoprecipitation-quantitative polymerase chain reaction, we found that METTL14 regulated Trib2 expression in an m6A-regulated manner, thereby activating the mitogen-activated protein kinase pathway and promoting corticospinal tract regeneration. Finally, we administered syringin, a stabilizer of METTL14, using molecular docking. Results confirmed that syringin can promote corticospinal tract regeneration and facilitate neurological recovery by stabilizing METTL14. Findings from this study reveal that m6A modification is involved in the regulation of corticospinal tract regeneration after spinal cord injury.

N6-methyladenosine (m6A) RNA methylation is crucially involved in the genesis and advancement of gastric cancer (GC) by controlling various pathobiological aspects including gene expression, signal transduction, metabolism, cell death, epithelial-mesenchymal transition, angiogenesis, and exosome function. Despite its importance, the exact mechanisms by which m6A modification influences GC biology remain inadequately explored. This review consolidates the latest advances in uncovering the mechanisms and diverse roles of m6A in GC and proposes new research and translational directions. Key regulators (writers, readers, and erasers) of m6A, such as METTL3/14/16 and WTAP, significantly affect cancer progression, anticancer immune response, and treatment outcomes. m6A modification also impacts immune cell infiltration and the tumor microenvironment, highlighting its potential as a diagnostic and prognostic marker. Interactions between m6A methylation and non-coding RNAs offer further novel insights into GC development and therapeutic targets. Targeting m6A regulators could enhance immunotherapy response, overcome treatment resistance, and improve oncological and clinical outcomes. Models based on m6A can precisely predict treatment response and prognosis in GC. Additional investigation is needed to fully understand the mechanisms of m6A methylation and its potential clinical applications and relevance (e.g., as precise markers for early detection, prediction of outcome, and response to therapy and as therapeutic targets) in GC. Future research should focus on in vivo studies, potential clinical trials, and the examination of m6A modification in other types of cancers.

N6-Methyladenosine (m6A) is a neuronal-enriched, reversible post-transcriptional modification that regulates RNA metabolism. The m6A-modified RNAs recruit various m6A-binding proteins that act as readers. Differential m6A methylation patterns are implicated in ischemic brain damage, yet the precise role of m6A readers in propagating post-stroke m6A signaling remains unclear. We presently evaluated the functional significance of the brain-enriched m6A reader YTHDF1, in post-stroke pathophysiology. Focal cerebral ischemia significantly increased YTHDF1 mRNA and protein expression in adult mice of both sexes. YTHDF1-/- male, but not female, mice subjected to transient middle cerebral artery occlusion (MCAO) showed worsened motor function recovery and increased infarction compared to sex-matched YTHDF1+/+ mice. YTHDF1-/- male, but not female, mice subjected to transient MCAO also showed significantly perturbed expression of genes related to inflammation, and increased infiltration of peripheral immune cells into the peri-infarct cortex, compared with sex-matched YTHDF1+/+ mice. Thus, this study demonstrates a sexual dimorphism of YTHDF1 in regulating post-ischemic inflammation and pathophysiology. Hence, post-stroke epitranscriptomic regulation might be sex-dependent.

Hepatoblastoma (HB) is a rare but predominant liver cancer in children, with few treatment choices in advanced stages. YWHAE is closely related to several human diseases and acts as a molecular scaffold for malignant transformation. However, whether YWHAE promotes HB development remains unknown. Conducting RNA and m6A sequencing on HB tissues, we found that YWHAE was upregulated and modified by N6-methyadenosine. Functionally, YWHAE promoted proliferation and inhibited cell death in HB by in vitro and in vivo studies. Mechanistically, METTL3-dependent m6A modification activated YWHAE mRNA expression, and the m6A reader IGF2BP2 recognized and bound to the m6A site on YWHAE mRNA, thereby enhancing the mRNA stability of YWHAE. Interestingly, RNA sequencing revealed that YWHAE knockdown was involved in regulating ferroptosis of HB cells by mediating SLC7A11 expression. Moreover, knockdown of YWHAE significantly increased the levels of lipid ROS and peroxides in HB cells, promoting the susceptibility of HB cells to ferroptosis. In summary, these findings illuminated the role of YWHAE in HB progression and uncovered its relevance to ferroptosis as a new therapeutic target for HB.

Circular RNAs (circRNAs) are non-coding RNAs with covalently closed loop structures that participate in various biological processes. However, the functions of many circRNAs remain unclear. Endothelial cell dysfunction, which involves abnormal ferroptosis, a unique form of regulated cell death, is a characteristic of various diseases. However, the mechanisms governing ferroptosis in endothelial cells are not fully understood. Here, we investigated the impact of a novel circRNA, circ-DAPK1, on ferroptosis in human umbilical vein endothelial cells (HUVECs) under high-glucose conditions. Our data showed that high-glucose conditions upregulate circ-DAPK1 expression in HUVECs. Overexpression of circ-DAPK1 induced ferroptosis in HUVECs, whereas depletion of circ-DAPK1 mitigated the ferroptosis triggered by high-glucose treatment. Inhibition of ferroptosis reversed the decrease in cell viability induced by high glucose or circ-DAPK1 overexpression. Using RNA immunoprecipitation analyses, we identified several ferroptosis-regulating proteins, including NAD(P)H dehydrogenase [quinone] 1 (NQO1) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2). Mechanistically, circ-DAPK1 interacts with NQO1, enhancing its ubiquitination and accelerating its degradation. NQO1 overexpression partially rescues HUVECs from high-glucose-induced ferroptosis. We also found that IGF2BP2 binds to the m6A site on circ-DAPK1. Depletion of IGF2BP2 in HUVECs reduced circ-DAPK1 expression and inhibited high-glucose-induced ferroptosis. These findings reveal the effects of the IGF2BP2-circ-DAPK1 axis in regulating ferroptosis in HUVECs under high-glucose conditions and extend our understanding of the mechanisms controlling ferroptosis in endothelial cells.

Oxaliplatin resistance poses a significant challenge in colorectal cancer (CRC) treatment. Recent studies have implicated CPSF6 in cancer progression and drug resistance, although its role in chemotherapy resistance and regulatory mechanisms is unclear. This study investigates CPSF6's involvement in oxaliplatin resistance in CRC and its regulation via m6A methylation by METTL3. We assessed CPSF6 expression in oxaliplatin-resistant (OxR) CRC cell lines (HT29-OxR and HCT116-OxR) compared to sensitive counterparts using qRT-PCR and Western blotting. CPSF6 was significantly upregulated in OxR cells, and its knockdown reduced cell viability, colony formation, and glycolytic activity while increasing apoptosis. m6A modification of CPSF6 mRNA was elevated in OxR cells, correlating with increased METTL3 expression. METTL3 knockdown decreased CPSF6 levels and m6A enrichment, enhancing mRNA degradation, while its overexpression stabilized CPSF6 mRNA, promoting resistance. Xenograft experiments showed that CPSF6 knockdown suppressed tumor growth and glycolysis. Thus, CPSF6 is identified as a mediator of oxaliplatin resistance in CRC, regulated by the METTL3/m6A axis, suggesting potential therapeutic targets to overcome resistance.

N6-methyladenosine (m6A) modification is the most abundant post-transcriptional modification of mRNAs and has been identified to play critical roles in ischemic stroke (IS). Herein, this study aimed to investigate the function and mechanism of Methyltransferase-like 14 (METTL14) methylase in cerebral IS. Murine BV-2 microglial cell OGD/R models and rat middle cerebral artery occlusion (MCAO) models were established to mimic IS-induced neuronal damage in vitro and brain injury in vivo. Levels of METTL14, Histone Deacetylase 3 (HDAC3) and cGAS-STING axis-related proteins were detected using qRT-PCR or western blotting. Cell proliferation and inflammation were assessed by CCK-8 assay, EdU assay and ELISA. Flow cytometry detected microglia polarization. Cell pyroptosis was analyzed by detecting related-protein markers by western blotting. The m6A modification was determined by methylated RNA immunoprecipitation assay. Brain injury was analyzed by evaluating infarct volume and neurologic score. METTL14 levels were higher in OGD/R-induced microglial cells, primary microglia and infarct brain tissues of rat MCAO models. Functionally, METTL14 silencing reversed OGD/R-induced proliferation inhibition, inflammation and pyroptosis in microglial cells and primary microglia in vitro, and ameliorated cerebral ischemic injury in rat MCAO models. Mechanistically, METTL14 induced HDAC3 m6A modification in an IGF2BP3-dependent manner, and could activate cGAS-STING pathway through HDAC3. Moreover, HDAC3 overexpression reversed the neuroprotective effects of METTL14 silencing. METTL14 silencing reversed ischemic stroke-induced brain injury by inducing HDAC3 m6A modification in an IGF2BP3-dependent mechanism, recommending a novel insight for ameliorating cerebral ischemic stroke.

Periodontitis, a chronic inflammatory condition, often results in gum tissue damage and can lead to tooth loss. This study explores the role of methyltransferase-like 3 (METTL3) in promoting osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) within an inflammatory microenvironment. An inflammatory environment was simulated in hPDLSCs using lipopolysaccharide (LPS). Both adipogenic and osteogenic differentiation capacities of hPDLSCs were assessed. In LPS-treated hPDLSCs, METTL3 was overexpressed, and alkaline phosphatase (ALP) staining was performed alongside measurements of ALP activity, pro-inflammatory cytokines, METTL3, miR-141-3p, pri-miR-141, Zinc finger E-box binding homeobox 1 (ZEB1), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN). N6-methyladenosine (m6A) and pri-miR-141 levels were quantified, and the binding of miR-141-3p to ZEB1 was analyzed. The results demonstrated that osteogenic differentiation in hPDLSCs was diminished under inflammatory conditions, coinciding with downregulated METTL3 expression. However, METTL3 overexpression enhanced osteogenic differentiation. METTL3 facilitated the conversion of pri-miR-141 into miR-141-3p via m6A modification, resulting in increased miR-141-3p levels, which in turn suppressed ZEB1 expression. Inhibition of miR-141-3p or overexpression of ZEB1 partially counteracted the positive effects of METTL3 on osteogenic differentiation. In conclusion, these findings suggest that METTL3-mediated m6A modification promotes osteogenic differentiation of hPDLSCs within an inflammatory microenvironment through the miR-141-3p/ZEB1 axis.

Enhancers, as distal cis-regulatory elements in the genome, have a pivotal influence on orchestrating precise gene expression. Enhancer RNAs (eRNAs), transcribed from active enhancer regions, are increasingly recognized as key regulators of transcription. N6-methyladenosine (m6A), the most plentiful internal modification in eukaryotic mRNAs, has garnered significant research interest in recent years. With advancements in high-throughput sequencing technologies, it has been established that m6A modifications are also present on eRNAs. An accumulative body of evidence demonstrates that aberrant enhancers, eRNAs, and m6A modifications are intimately connected with carcinoma onset, progression, invasion, metastasis, treatment response, drug resistance, and prognosis. However, the underlying molecular mechanisms governing m6A modification of eRNAs in cancer remain elusive. Here, we review and synthesize current understanding of the regulatory roles of enhancers, eRNAs, and m6A modifications in cancer. Furthermore, we investigate the possible roles of eRNAs m6A modification in tumorigenesis based on existing literature, offering novel perspectives and directions for future research on epigenetic regulatory mechanisms in cancer cells.