m6A-dependent Manner Research Articles

M6A methyltransferase METTL3 programs CD4+ T-cell activation and effector T-cell differentiation in systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disorder in which excessive CD4+ T-cell activation and imbalanced effector T-cell differentiation play critical roles. Recent studies have implied a potential association between posttranscriptional N6-methyladenosine (m6A) modification and CD4+ T-cell-mediated humoral immunity. However, how this biological process contributes to lupus is not well understood. In this work, we investigated the role of the m6A methyltransferase like 3 (METTL3) in CD4+ T-cell activation, differentiation, and SLE pathogenesis both in vitro and in vivo.MethodsThe expression of METTL3 was knocked down and METTL3 enzyme activity was inhibited using siRNA and catalytic inhibitor, respectively. In vivo evaluation of METTL3 inhibition on CD4+ T-cell activation, effector T-cell differentiation, and SLE pathogenesis was achieved using a sheep red blood cell (SRBC)-immunized mouse model and a chronic graft versus host disease (cGVHD) mouse model. RNA-seq was performed to identify pathways and gene signatures targeted by METTL3. m6A RNA-immunoprecipitation qPCR was applied to confirm the m6A modification of METTL3 targets.ResultsMETTL3 was defective in the CD4+ T cells of SLE patients. METTL3 expression varied following CD4+ T-cell activation and effector T-cell differentiation in vitro. Pharmacological inhibition of METTL3 promoted the activation of CD4+ T cells and influenced the differentiation of effector T cells, predominantly Treg cells, in vivo. Moreover, METTL3 inhibition increased antibody production and aggravated the lupus-like phenotype in cGVHD mice. Further investigation revealed that catalytic inhibition of METTL3 reduced Foxp3 expression by enhancing Foxp3 mRNA decay in a m6A-dependent manner, hence suppressing Treg cell differentiation.ConclusionIn summary, our findings demonstrated that METTL3 was required for stabilizing Foxp3 mRNA via m6A modification to maintain the Treg differentiation program. METTL3 inhibition contributed to the pathogenesis of SLE by participating in the activation of CD4+ T cells and imbalance of effector T-cell differentiation, which could serve as a potential target for therapeutic intervention in SLE.

FTO-mediated m6A demethylation of pri-miR-3591 alleviates osteoarthritis progression

ObjectivesIncreasing evidence have demonstrated the N6-methyladenosine (m6A) plays critical roles in osteoarthritis (OA) progression, but the role of m6A in OA has not been completely illuminated. Herein, we investigated the function and underlying mechanism of m6A demethylase fat mass and obesity-associated protein (FTO) in OA progression.Materials and methodsThe FTO expression was detected in mice OA cartilage tissues and lipopolysaccharide (LPS)-stimulated chondrocytes. Gain-of-function assays was used to evaluate the role of FTO in OA cartilage injury in vitro and in vivo. The miRNA-sequencing, RNA-binding protein immunoprecipitation (RIP), luciferase reporter assay, and in vitro pri-miRNA processing assays were conducted to confirm that FTO modulated the pri-miR-3591 process in an m6A-dependent manner and then the binding sites of miR-3591-5p with PRKAA2.ResultsFTO was outstandingly downregulated in LPS-stimulated chondrocytes and OA cartilage tissues. FTO overexpression enhanced the proliferation, suppressed apoptosis, and decreased degradation of extracellular matrix in LPS-induced chondrocytes, whereas FTO knockdown contributed to the opposite effects. In vivo animal experiments showed that FTO overexpression markedly alleviated OA mice cartilage injury. Mechanically, FTO-mediated m6A demethylation of pri-miR-3591 leaded to a maturation block of miR-3591-5p, which relieved the inhibitory effect of miR-3591-5p on PRKAA2 and then promoted the increase of PRKAA2, thereby alleviating OA cartilage damage.ConclusionsOur results attested that FTO alleviated the OA cartilage damage by mediating FTO/miR-3591-5p/PRKAA2 axis, which provided fresh insights into the therapeutic strategies for OA.

SH3BP5-AS1/IGF2BP2/VDAC2 Axis Promotes the Apoptosis and Ferroptosis of Bladder Cancer Cells.

Bladder cancer (BC) is the most common malignant tumor in the urinary system with a high incidence, imposing a burden on the healthcare system worldwide. The participation of long non-coding RNAs (lncRNAs) in BC has attracted increasing attention. The aim in the current study was to explore the potential mechanism involving SH3BP5-AS1 in modulating BC cell proliferation, apoptosis and ferroptosis. qPCR and WB analysis measured the expression of RNAs and proteins. Functional and mechanism experiments were performed to investigate RNA impacts on cell proliferation, apoptosis and ferroptosis, and explore the correlation between RNA and protein expression. SH3BP5-AS1 was down-regulated in BC cells, and SH3BP5-AS1 overexpression could inhibit BC cell proliferation but facilitate the cell apoptosis. SH3BP5-AS1 was also found to facilitate the ferroptosis of BC cells. Additionally, SH3BP5-AS1 was confirmed to recruit IGF2BP2 to regulate VDAC2 expression in the m6A-dependent manner. VDAC2 was detected to be down-regulated in BC cells and was verified to inhibit BC cell growth. Moreover, it was indicated from rescue assays that SH3BP5-AS1 could modulate VDAC2 expression to promote the ferroptosis of BC cells. SH3BP5-AS1 could affect BC cell proliferation, apoptosis and ferroptosis via IGF2BP2/VDAC2, providing a novel molecular perspective for understanding BC.

LRPPRC facilitates tumor progression and immune evasion through upregulation of m6A modification of PD-L1 mRNA in hepatocellular carcinoma.

LRPPRC is a newly discovered N6-methyladenosine (m6A) modification reader, which potentially affects hepatocellular carcinoma (HCC) progression. PD-L1 in tumor cells is essential for tumor immune evasion. This work investigated the LRPPRC-mediated m6A-modification effect on PD-L1 mRNA and immune escape in HCC. Expression and clinical implication of LRPPRC and PD-L1 were measured in human HCC cohorts. The influence of LRPPRC on malignant behaviors of HCC cells was investigated through in vitro assays and xenograft tumor murine models. The posttranscriptional mechanism of LRPPRC on PD-L1 and anti-tumor immunity was elucidated in HCC cells via RIP, MeRIP-qPCR, RNA stability, immunohistochemical staining, and so forth. LRPPRC exhibited the notable upregulated in human HCC tissues, which was in relation to advanced stage and worse overall survival and disease-free survival. Impaired proliferative capacity and G2/M phage arrest were found in LRPPRC-knockout cells, with increased apoptotic level, and attenuated migratory and invasive abilities. In HCC patients and murine models, LRPPRC presented a positive interaction with PD-L1, with negative associations with CD8+, and CD4+ T-cell infiltrations and chemokines CXCL9, and CXCL10. LRPPRC loss downregulated the expression of PD-L1 and its m6A level in HCC cells. Moreover, LRPPRC suppression mitigated tumor growth in murine models and improved anti-tumor immunity and immune infiltration in tumors. This work unveiled that LRPPRC may posttranscriptionally upregulate PD-L1 partially with an m6A-dependent manner for heightening mRNA stabilization of PD-L1 and provided a new mechanism for m6A regulator-mediated immunosuppression in HCC.

WTAP contributes to the tumorigenesis of osteosarcoma via modulating ALB in an m6A‐dependent manner

Osteosarcoma (OS) is a prevalent bone malignancy mainly occurred in adolescents. WTAP/N6-methyladenosine (m6A) modification is confirmed to be involved in OS progression. This study is conducted to bring some novel insights to the action mechanism of WTAP/m6A under the hidden pathogenesis of OS. qRT-PCR was executed to evaluate the expression levels of WTAP and ALB. ALB protein level in OS cells was measured by western blotting. The content of m6A in total RNA was assessed by m6A quantification assay. Me-RIP, dual luciferase reporter, and mRNA stability assays confirmed the target relationship of WTAP with ALB. With the use of the wound healing, CCK-8, and transwell invasion assays, the functional relationship between WTAP and ALB in OS cells was confirmed. The influences of WTAP on tumor growth in vivo were performed in the xenograft model of mouse. WTAP was increased but ALB was diminished in OS tissues and/or cell lines. WTAP modulated ALB expression in an m6A-dependent manner. Silencing of WTAP retarded the development of OS via inhibiting cell viability, migration, invasion, and tumor growth. Knockdown of ALB exerted the opposite effects on OS progression. Additionally, ALB deficiency partially eliminated the inhibiting effects of WTAP silencing on cellular processes in OS. This is the first report to clarify the interaction of WTAP/m6A with ALB in OS progression. These experimental data to some extent broadened the horizons of WTAP/m6A in the development of OS.

Kinesin Family Member C1 Overexpression Exerts Tumor-Promoting Properties in Head and Neck Squamous Cell Carcinoma via the Rac1/Wnt/β-catenin Pathway

Kinesin family member C1 (KIFC1) is a kinesin-14 motor protein, and its abnormal upregulation promotes the malignant behavior of cancer cells. N6-methyladenosine (m6A) RNA methylation is a common modification of eukaryotic messenger RNA and affects RNA expression. In this study, we explored how KIFC1 regulated head and neck squamous cell carcinoma (HNSCC) tumorigenesis and how m6A modification affected KIFC1 expression. A bioinformatics analysis was performed to screen for genes of interest, and in vitro and in vivo studies were carried out to investigate the function and mechanism of KIFC1 in HNSCC tissues. We observed that the expression of KIFC1 in HNSCC tissues was significantly higher than that in normal or adjacent normal tissues. Patients with cancer with higher KIFC1 expression have a lower tumor differentiation status. Demethylase alkB homolog 5, a cancer-promoting factor in HNSCC tissues, could interact with KIFC1 messenger RNA and posttranscriptionally activate KIFC1 through m6A modification. KIFC1 downregulation suppressed HNSCC cell growth and metastasis in vivo and in vitro. However, overexpression of KIFC1 promoted these malignant behaviors. We demonstrated that KIFC1 overexpression activated the oncogenic Wnt/β-catenin pathway. KIFC1 interacted with the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1) at the protein level and increased its activity. The Rho GTPase Rac1 was indicated to be an upstream activator of the Wnt/β-catenin signaling pathway, and its Rac1 inhibitor, NSC-23766, treatment reversed the effects caused by KIFC1 overexpression. Those observations demonstrate that abnormal expression of KIFC1 may be regulated by demethylase alkB homolog 5 in an m6A-dependent manner and promote HNSCC progression via the Rac1/Wnt/β-catenin pathway.

N6-methyladenosine reader YTHDF1 regulates the proliferation and migration of airway smooth muscle cells through m6A/cyclin D1 in asthma.

Asthma is a chronic inflammatory respiratory disease, which is involved in multiple pathologic molecular mechanisms and presents a huge challenge to clinic nursing. Emerging evidence suggests that N6-methyladenosine (m6A) plays critical roles in respiratory system disease. Thus, present work tried to investigate the functions of m6A reader YTHDF 1 in asthma. The results indicated that YTHDF1 significantly upregulated in platelet-derived growth factor (PDGF) induced airway smooth muscle cells (ASMCs). Functionally, overexpression of YTHDF1 promoted the proliferation and migration of ASMCs, while YTHDF1 knockdown repressed the proliferation and migration. Mechanistically, there was a m6A modification site on cyclin D1 RNA (CCND1 genome) and YTHDF1 combined with cyclin D1 mRNA, thereby enhancing its mRNA stability via m6A-dependent manner. Collectively, these findings reveal a novel axis of YTHDF1/m6A/cyclin D1 in asthma's airway remodeling, which may provide novel therapeutic strategy for asthma.

METTL3-Mediated LncRNA EN_42575 m6A Modification Alleviates CPB2 Toxin-Induced Damage in IPEC-J2 Cells.

Long non-coding RNAs (lncRNAs) modified by n6-methyladenosine (m6A) have been implicated in the development and progression of several diseases. However, the mechanism responsible for the role of m6A-modified lncRNAs in Clostridium perfringens type C piglet diarrhea has remained largely unknown. We previously developed an in vitro model of CPB2 toxin-induced piglet diarrhea in IPEC-J2 cells. In addition, we previously performed RNA immunoprecipitation sequencing (MeRIP-seq), which demonstrated lncRNA EN_42575 as one of the most regulated m6A-modified lncRNAs in CPB2 toxin-exposed IPEC-J2 cells. In this study, we used MeRIP-qPCR, FISH, EdU, and RNA pull-down assays to determine the function of lncRNA EN_42575 in CPB2 toxin-exposed IPEC-J2 cells. LncRNA EN_42575 was significantly downregulated at different time points in CPB2 toxin-treated cells. Functionally, lncRNA EN_42575 overexpression reduced cytotoxicity, promoted cell proliferation, and inhibited apoptosis and oxidative damage, whereas the knockdown of lncRNA EN_42575 reversed these results. Furthermore, the dual-luciferase analysis revealed that METTL3 regulated lncRNA EN_42575 expression in an m6A-dependent manner. In conclusion, METTL3-mediated lncRNA EN_42575 exerted a regulatory effect on IPEC-J2 cells exposed to CPB2 toxins. These findings offer novel perspectives to further investigate the function of m6A-modified lncRNAs in piglet diarrhea.

METTL3 promotes proliferation of goat endometrial epithelial cells by regulating CTGF in an m6A-dependent manner.

N6-methyladenosine (m6A), an epigenetic modification on RNAs, plays an important role in many physiological and pathological processes. However, the involvement of m6A in goat uterus during early pregnancy remains largely unknown. In this study, we found that the total m6A level was increasing in goat uterus as early pregnancy progressed. Methyltransferase-like 3 (METTL3) is a core catalytic subunit of the m6A methyltransferase. We thus determined the expression and regulation of METTL3 in goat uterus. METTL3 was highly expressed in the luminal and glandular epithelia from day 16 (D16) to D25 of pregnancy. And it could be up-regulated by estrogen and progesterone in goat uterus and primary endometrial epithelial cells (EECs). In EECs, knockdown or overexpression of METTL3 resulted in a significant decrease or increase of cell proliferation, respectively. METTL3 knockdown reduced the m6A level of not only total RNA but also connective tissue growth factor (CTGF) mRNA. Luciferase assay suggested that METTL3 might target the potential m6A sites in the 3'untranslated region (3'UTR) of CTGF mRNA. Moreover, METTL3 positively regulated CTGF expression, and CTGF knockdown significantly counteracted the promoting effect of METTL3 overexpression on EEC proliferation. Collectively, METTL3 is dynamically expressed in goat uterus and can affect EEC proliferation by regulating CTGF in an m6A-dependent manner. Our results will lay a foundation for further studying the crucial mechanism of METTL3-mediated m6A modification in goat uterus during early pregnancy.

METTL3 potentiates osteogenic differentiation of bone marrow mesenchymal stem cells via IGF2BP1/m6A/RUNX2.

Maxillofacial bone defect is a critical obstacle for maxillofacial tumors and periodontal diseases. The osteogenic differentiation of bone marrow mesenchymal stem cells BMSCs is critical for maxillofacial osteogenesis and functional reconstruction. Here, our study focused on the functions and mechanism of N6-methyladenosine during BMSCs osteogenic differentiation BMSCs. Biofunctions of BMSCs were detected using ALP activity and alizarin red S staining assays. The molecular interaction within RNA/protein was identified by RNA immunoprecipitation and/or methylation immunoprecipitation. Results indicated that m6A 'writer' METTL3 upregulated during the osteogenic differentiation of BMSCs upon osteogenic induction. Functionally, assays' results revealed that METTL3 overexpression promoted the osteogenic differentiation of BMSC, while METTL3 knockdown repressed the osteogenic differentiation. Mechanistically, results revealed that RUNX2 mRNA was a m6A-methylated target by METTL3 at its 3'-UTR. Moreover, m6A reader IGF2BP1 recognized the m6A site on RUNX2 mRNA to enhance its stability. In conclusion, our findings revealed the novel roles of METTL3 in BMSCs osteogenic differentiation via the IGF2BP1/m6A/RUNX2 signaling axis of m6A-dependent manner, providing a potential therapeutic target for maxillofacial bone defects treatment.

Adipocyte YTH N(6)-methyladenosine RNA-binding protein 1 protects against obesity by promoting white adipose tissue beiging in male mice

Obesity, one of the most serious public health issues, is caused by the imbalance of energy intake and energy expenditure. N(6)-methyladenosine (m6A) RNA modification has been recently identified as a key regulator of obesity, while the detailed mechanism is elusive. Here, we find that YTH RNA binding protein 1 (YTHDF1), an m6A reader, acts as an essential regulator of white adipose tissue metabolism. The expression of YTHDF1 decreases in adipose tissue of male mice fed a high-fat diet. Adipocyte-specific Ythdf1 deficiency exacerbates obesity-induced metabolic defects and inhibits beiging of inguinal white adipose tissue (iWAT) in male mice. By contrast, male mice with WAT-specific YTHDF1 overexpression are resistant to obesity and shows promotion of beiging. Mechanistically, YTHDF1 regulates the translation of diverse m6A-modified mRNAs. In particular, YTHDF1 facilitates the translation of bone morphogenetic protein 8b (Bmp8b) in an m6A-dependent manner to induce the beiging process. Here, we show that YTHDF1 may be an potential therapeutic target for the management of obesity-associated diseases.

U-shaped association between serum IGF2BP3 and T2DM: A cross-sectional study in Chinese population.

To clarify the expression of N6-methyladenosine (m6 A) modulators involved in the pathogenesis of type 2 diabetes mellitus (T2DM). We further explored the association of serum insulin-like growth factor 2 mRNA-binding proteins 3 (IGF2BP3) levels and odds of T2DM in a high-risk population. The gene expression data set GSE25724 was obtained from the Gene Expression Omnibus, and a cluster heatmap was generated by using the R package ComplexHeatmap. Differential expression analysis for 13 m6 A RNA methylation regulators between nondiabetic controls and T2DM subjects was performed using an unpaired t test. A cross-sectional design, including 393 subjects (131 patients with newly diagnosed T2DM, 131 age- and sex-matched subjects with prediabetes, and 131 healthy controls), was carried out. The associations between serum IGF2BP3 concentrations and T2DM were modeled by restricted cubic spline and logistic regression models. Two upregulated (IGF2BP2 and IGF2BP3) and 5 downregulated (methyltransferase-like 3 [METTL3], alkylation repair homolog protein 1 [ALKBH1], YTH domain family 2 [YTHDF2], YTHDF3, and heterogeneous nuclear ribonucleoprotein [HNRNPC]) m6 A-related genes were found in islet samples of T2DM patients. A U-shaped association existed between serum IGF2BP3 levels and odds of T2DM according to cubic natural spline analysis models, after adjustment for body mass index, waist circumference, diastolic blood pressure, total cholesterol, and triglyeride. Multivariate logistic regression showed that progressively higher odds of T2DM were observed when serum IGF2BP3 levels were below 0.62 ng/mL (odds ratio 3.03 [95% confidence interval 1.23-7.47]) in model 4. Seven significantly altered m6 A RNA methylation genes were identified in T2DM. There was a U-shaped association between serum IGF2BP3 levels and odds of T2DM in the general Chinese adult population. This study provides important evidence for further examination of the role of m6 A RNA methylation, especially serum IGF2BP3 in T2DM risk assessment.

Global loss of cellular m6A RNA methylation following infection with different SARS-CoV-2 variants.

Insights into host-virus interactions during SARS-CoV-2 infection are needed to understand COVID-19 pathogenesis and may help to guide the design of novel antiviral therapeutics. N 6-Methyladenosine modification (m6A), one of the most abundant cellular RNA modifications, regulates key processes in RNA metabolism during stress response. Gene expression profiles observed postinfection with different SARS-CoV-2 variants show changes in the expression of genes related to RNA catabolism, including m6A readers and erasers. We found that infection with SARS-CoV-2 variants causes a loss of m6A in cellular RNAs, whereas m6A is detected abundantly in viral RNA. METTL3, the m6A methyltransferase, shows an unusual cytoplasmic localization postinfection. The B.1.351 variant has a less-pronounced effect on METTL3 localization and loss of m6A than did the B.1 and B.1.1.7 variants. We also observed a loss of m6A upon SARS-CoV-2 infection in air/liquid interface cultures of human airway epithelia, confirming that m6A loss is characteristic of SARS-CoV-2-infected cells. Further, transcripts with m6A modification are preferentially down-regulated postinfection. Inhibition of the export protein XPO1 results in the restoration of METTL3 localization, recovery of m6A on cellular RNA, and increased mRNA expression. Stress granule formation, which is compromised by SARS-CoV-2 infection, is restored by XPO1 inhibition and accompanied by a reduced viral infection in vitro. Together, our study elucidates how SARS-CoV-2 inhibits the stress response and perturbs cellular gene expression in an m6A-dependent manner.

M6A demethylase ALKBH5 attenuates doxorubicin-induced cardiotoxicity via posttranscriptional stabilization of Rasal3

The clinical application of anthracyclines such as doxorubicin (DOX) is limited due to their cardiotoxicity. N6-methyladenosine (m6A) plays an essential role in numerous biological processes. However, the roles of m6A and m6A demethylase ALKBH5 in DOX-induced cardiotoxicity (DIC) remain unclear. In this research, DIC models were constructed using Alkbh5-knockout (KO), Alkbh5-knockin (KI), and Alkbh5-myocardial-specific knockout (ALKBH5flox/flox, αMyHC-Cre) mice. Cardiac function and DOX-mediated signal transduction were investigated. As a result, both Alkbh5 whole-body KO and myocardial-specific KO mice had increased mortality, decreased cardiac function, and aggravated DIC injury with severe myocardial mitochondrial damage. Conversely, ALKBH5 overexpression alleviated DOX-mediated mitochondrial injury, increased survival, and improved myocardial function. Mechanistically, ALKBH5 regulated the expression of Rasal3 in an m6A-dependent manner through posttranscriptional mRNA regulation and reduced Rasal3 mRNA stability, thus activating RAS3, inhibiting apoptosis through the RAS/RAF/ERK signaling pathway, and alleviating DIC injury. These findings indicate the potential therapeutic effect of ALKBH5 on DIC.

Ythdf2 promotes pulmonary hypertension by suppressing Hmox1-dependent anti-inflammatory and antioxidant function in alveolar macrophages

Pulmonary hypertension (PH) is a devastating disease characterized by irreversible pulmonary vascular remodeling (PVR) that causes right ventricular failure and death. The early alternative activation of macrophages is a critical event in the development of PVR and PH, but the underlying mechanisms remain elusive. Previously we have shown that N6-methyladenosine (m6A) modifications of RNA contribute to phenotypic switching of pulmonary artery smooth muscle cells and PH. In the current study, we identify Ythdf2, an m6A reader, as an important regulator of pulmonary inflammation and redox regulation in PH. In a mouse model of PH, the protein expression of Ythdf2 was increased in alveolar macrophages (AMs) during the early stages of hypoxia. Mice with a myeloid specific knockout of Ythdf2 (Ythdf2Lyz2 Cre) were protected from PH with attenuated right ventricular hypertrophy and PVR compared to control mice and this was accompanied by decreased macrophage polarization and oxidative stress. In the absence of Ythdf2, heme oxygenase 1 (Hmox1) mRNA and protein expression were significantly elevated in hypoxic AMs. Mechanistically, Ythdf2 promoted the degradation of Hmox1 mRNA in a m6A dependent manner. Furthermore, an inhibitor of Hmox1 promoted macrophage alternative activation, and reversed the protection from PH seen in Ythdf2Lyz2 Cre mice under hypoxic exposure. Together, our data reveal a novel mechanism linking m6A RNA modification with changes in macrophage phenotype, inflammation and oxidative stress in PH, and identify Hmox1 as a downstream target of Ythdf2, suggesting that Ythdf2 may be a therapeutic target in PH.

M6A-modified circRNA MYO1C participates in the tumor immune surveillance of pancreatic ductal adenocarcinoma through m6A/PD-L1 manner

Emerging evidence indicates the critical roles of N6-methyladenosine (m6A) modification in human cancers. Herein, our work reported that a novel m6A-modified circRNA from the MYO1C gene, circMYO1C, upregulated in the pancreatic ductal adenocarcinoma (PDAC). Our findings demonstrated that circMYO1C is highly expressed in PDAC tissues. Functionally, circMYO1C promoted the proliferation and migration of PDAC cells in vitro and its silencing reduced the tumor growth in vivo. Mechanistically, circMYO1C cyclization was mediated by m6A methyltransferase METTL3. Moreover, methylated RNA immunoprecipitation sequencing (MeRIP-seq) unveiled the remarkable m6A modification on PD-L1 mRNA. Moreover, circMYO1C targeted the m6A site of PD-L1 mRNA to enhance its stability by cooperating with IGF2BP2, thereby accelerating PDAC immune escape. In conclusion, these findings highlight the oncogenic role of METTL3-induced circMYO1C in PDAC tumorigenesis via an m6A-dependent manner, inspiring a novel strategy to explore PDAC epigenetic therapy.

O-GlcNAcylation of YTHDF2 promotes HBV-related hepatocellular carcinoma progression in an N6-methyladenosine-dependent manner

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), but its pathogenic mechanism remains to be explored. The RNA N6-methyladenosine (m6A) reader, YTH (YT521-B homology) domain 2 (YTHDF2), plays a critical role in the HCC progression. However, the function and regulatory mechanisms of YTHDF2 in HBV-related HCC remain largely elusive. Here, we discovered that YTHDF2 O-GlcNAcylation was markedly increased upon HBV infection. O-GlcNAc transferase (OGT)-mediated O-GlcNAcylation of YTHDF2 on serine 263 enhanced its protein stability and oncogenic activity by inhibiting its ubiquitination. Mechanistically, YTHDF2 stabilized minichromosome maintenance protein 2 (MCM2) and MCM5 transcripts in an m6A-dependent manner, thus promoting cell cycle progression and HBV-related HCC tumorigenesis. Moreover, targeting YTHDF2 O-GlcNAcylation by the OGT inhibitor OSMI-1 significantly suppressed HCC progression. Taken together, our findings reveal a new regulatory mechanism for YTHDF2 and highlight an essential role of YTHDF2 O-GlcNAcylation in RNA m6A methylation and HCC progression. Further description of the molecular pathway has the potential to yield therapeutic targets for suppression of HCC progression due to HBV infection.

YTHDF2 controls hexavalent chromium-induced mitophagy through modulating Hif1α and Bnip3 decay via the m6A/mRNA pathway in spermatogonial stem cells/progenitors

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, and SSC homeostasis is essential for lifelong male fertility. Currently, environmental pollution remains one of the factors affecting human reproductive health. Chromium is a prevalent metal element, and excessive exposure to hexavalent chromium (Cr (VI)) can cause male reproductive disorders. Nevertheless, the toxic effects of Cr (VI) on SSCs and the underlying mechanisms remain incompletely understood. Here, we showed that Cr (VI) exposure triggered mitophagy in mouse SSCs/progenitors in a time-dependent manner. Concurrently, Cr (VI) treatment caused reactive oxygen species (ROS) accumulation and activated the HIF1α-mediated BNIP3 expression to trigger mitophagy. In addition, Cr (VI) exposure significantly decreased the level of m6A modification. Further, we identified that YTHDF2 regulated the stability of Bnip3 and Hif1α mRNAs in an m6A-dependent manner, which was involved in Cr (VI)-induced mitophagy. Collectively, our study not only expands the mechanisms for Cr (VI)-caused male reproductive toxicity, but also provides pharmacological targets for prevention and treatment of Cr (VI)-induced male fertility impairment.

Inhibition of YTHDF1 prevents hypoxia-induced pulmonary artery smooth muscle cell proliferation by regulating Foxm1 translation in an m6A-dependent manner

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by pulmonary vascular remodeling. It refers to the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs), and hypoxia is an important risk factor for this progression. The present study aims to investigate the role of YTHDF1 in the regulation of hypoxic PASMC proliferation and the underlying mechanism. Human PASMCs were transfected with si-YTHDF1/2/3 followed by treatment of hypoxia, and the PASMC proliferation and Foxm1 expression were detected. Through RNA pull-down, RNA immunoprecipitation, and protein synthesis assay, the mechanism of YTHDF1 regulating Foxm1 was explored. Next, Foxm1 was inhibited by thiostrepton, and cell proliferation was detected. In vivo, mice received a tail vein injection of adenovirus containing si-YTHDF1 and were exposed to hypoxia treatment. Pulmonary vascular changes, right ventricular systolic pressure (RVSP), and genes involving proliferation were analyzed. YTHDF1 silencing reduced more hypoxic PASMC proliferation and Foxm1 protein level than YTHDF2/3 silencing. Mechanical results showed that YTHDF1 interacted with Foxm1 mRNA and up-regulated Foxm1 protein level by enhancing the translation efficiency in an m6A-dependent manner. Furthermore, YTHDF1 facilitated hypoxic PASMC proliferation and proliferation marker expressions through up-regulation of Foxm1 in an m6A-dependent manner. In vivo, the YTHDF1 silencing alleviated pulmonary vascular changes and fibrosis, reduced RVSP, inhibited the interaction of YTHDF1 and Foxm1, and reduced proliferation marker levels, as compared to the PAH group. In conclusion, YTHDF1 silencing inhibits hypoxic PASMC proliferation by regulating Foxm1 translation in an m6A-dependent manner.

METTL3 enhances the effect of YTHDF1 on NEDD1 mRNA stability by m6A modification in diffuse large B-cell lymphoma cells.

Diffuse large B-cell lymphoma (DLBCL) remains the most frequent subpopulation of lymphoma, and N6-methyladenosine (m6A) was implicated in the DLBCL progression. Herein, we sought to decipher the m6A-asociated mechanism of NEDD1 in DLBCL development. The NEDD1 expression profile in DLBCL was assessed by quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot. NEDD1 was artificially downregulated or upregulated in DLBCL cells, followed by EdU, Transwell assays and flow cytometry. The Hedgehog pathway activity was assayed by a dual-luciferase assay. The m6A methylation of NEDD1 in DLBCL was assessed by meRIP-qPCR, and the regulatory mechanism of METTL3 on NEDD1 was validated. The LDH assay was conducted to examine the impact of CD8+ T cells on DLBCL cells. The DLBCL cells were administrated into mice to evaluate the tumorigenic activity and ki-67 activity in tumor tissues. NEDD1 was overexpressed in DLBCL. Depletion of NEDD1 inhibited the aggressiveness of SU-DHL-8 and OCI-LY1 cells, whereas overexpression of NEDD1 expedited the aggressiveness of SU-DHL-8 and OCI-LY1 cells. METTL3 promoted NEDD1 translation in an m6A-dependent manner via YTHDF1. Depletion of METTL3 inhibited SU-DHL-8 and OCI-LY1 cell activity through regulation of NEDD1. NEDD1 reversed the repressive effect of METTL3 loss on the aggressiveness of SU-DHL-8 and OCI-LY1 cells. NEDD1 activated the Hedgehog signaling to promote immune escape of DLBCL. METTL3 promotes translation of NEDD1 via YTHDF1-depedndent m6A modification, thereby activating the Hedgehog signaling pathway to promote immune escape of DLBCL cells.