YTHDF1 Expression Research Articles

YTHDF2 promotes gastric cancer progression and enhances chemoradiotherapy resistance.

The role of YTHDF2 in gastric cancer (GC) is controversial. Due to the limitations of technical difficulty and experimental period, research on completely knocking out YTHDF2 is rare. Therefore, further investigations are still needed to clarify the YTHDF2's clinical significance and biological function in GC. To carry out the investigation, an analysis was performed on the expression levels of YTHDF2 in both publicly available databases and samples obtained from patients with gastric cancer. Based on the complete knockout of YTHDF2 using the CRISPR-Cas9 system, in vivo and in vitro experiments were conducted to analyze the effects of YTHDF2 on tumor formation, radiotherapy and chemoradiotherapy resistance in GC. Our investigation revealed an increase in YTHDF2 levels in GC tissues, which was found to be associated with a negative prognosis. Under hypoxic conditions, high expression of YTHDF2 enhanced the invasion of gastric cancer cells, and high expression of YTHDF2 was associated with HIF-1a. YTHDF2 facilitated gastric cancer cell growth in vitro and in vivo. Moreover, the results of the present study demonstrated that YTHDF2 mediated the expression of CyclinD1 and stability of CyclinD1 mRNA. CyclinD1 knockdown inhibited YTHDF2-mediated GC cell proliferation whereas CyclinD1 overexpression ameliorated YTHDF2 knockdown-induced inhibition of GC progression. Furthermore, YTHDF2 also promoted resistance to DDP and CTX chemotherapy, along with radiotherapy treatment for GC cells. The findings suggested that YTHDF2 expression accelerated GC progression through a potential mechanism involving CyclinD1 expression, and enhanced chemoradiotherapy resistance. This indicated that YTHDF2 could be a promising prognostic biomarker and therapeutic target for individuals diagnosed with GC.

YTHDF1 promotes the viability and self‑renewal of glioma stem cells by enhancing LINC00900 stability.

YTHDF1, an N6‑methyladenosine (m6A)‑binding protein, is significantly upregulated in glioma tissues. The present study investigated the molecular mechanism underlying the regulatory effects of YTHDF1 on the viability, invasion and self‑renewal of glioma stem cells (GSCs). Glioma and normal brain tissues were collected, and reverse transcription‑quantitative PCR and western blotting were used to measure the gene and protein expression levels, respectively. Methylated RNA immunoprecipitation‑PCR was used to assess the m6A modification level of the target gene. Subsequently GSCs were induced, and YTHDF1 and LINC00900 gene regulation was carried out using lentiviral infection. The viability, invasion and self‑renewal of GSCs were assessed by Cell Counting Kit‑8, Transwell and sphere formation assays, respectively. Binding between YTHDF1 and LINC00900 was verified by RNA immunoprecipitation and RNA pull‑down assays. The targeted binding of microRNA (miR)‑1205 to the LINC00900/STAT3 3'‑UTR was verified using a luciferase reporter assay. The results revealed that YTHDF1 and LINC00900 expression levels were significantly upregulated in glioma tissues, and a high m6A modification level in LINC00900 transcripts was detected in glioma tissues. Overexpression of YTHDF1 promoted GSC viability, invasion and self‑renewal, whereas knockdown of YTHDF1 had the opposite effects. In addition, YTHDF1 maintained the stability of LINC00900 and upregulated its expression through binding to it, thereby promoting GSC viability, invasion and self‑renewal. Furthermore, LINC00900 promoted GSC viability, invasion, self‑renewal and tumor growth by regulating the miR‑1205/STAT3 axis. In conclusion, YTHDF1 promotes GSC viability and self‑renewal by regulating the LINC00900/miR‑1205/STAT3 axis.

Epstein-Barr virus suppresses N6-methyladenosine modification of TLR9 to promote immune evasion

Epstein-Barr virus (EBV) is a human tumor virus associated with a variety of malignancies, including nasopharyngeal carcinoma, gastric cancers, and B-cell lymphomas. N6-methyladenosine (m6A) modifications modulate a wide range of cellular processes and participate in the regulation of virus-host cell interactions. Here, we discovered that EBV infection downregulates toll-like receptor 9 (TLR9) m6A modification levels and thus inhibits TLR9 expression. TLR9 has multiple m6A modification sites. Knockdown of METTL3, an m6A "writer", decreases TLR9 protein expression by inhibiting its mRNA stability. Mechanistically, Epstein-Barr nuclear antigen 1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway. Additionally, YTHDF1 was identified as an m6A "reader" of TLR9, enhancing TLR9 expression by promoting mRNA translation in an m6A -dependent manner, which suggests that EBV inhibits TLR9 translation by "hijacking" host m6A modification mechanism. Using the METTL3 inhibitor STM2457 inhibits TLR9-induced B cell proliferation and immunoglobulin secretion, and opposes TLR9-induced immune responses to assist tumor cell immune escape. In clinical lymphoma samples, the expression of METTL3, YTHDF1, and TLR9 was highly correlated with immune cells infiltration. This study reveals a novel mechanism that EBV represses the important innate immunity molecule TLR9 through modulating the host m6A modification system.

Dexmedetomidine suppressed the biological behavior of RAW264.7 cells treated with LPS by down-regulating HOTAIR

BackgroundPrevious studies have revealed dexmedetomidine have potential protective effects on vital organs by inhibiting the release of inflammatory cytokines. To investigate the effects of dexmedetomidine on sepsis, especially in the initial inflammatory stage of sepsis. RAW264.7 cells were used as the cell model in this study to elucidate the underlying mechanisms. MethodsIn this study, we conducted several assays to investigate the mechanisms of dexmedetomidine and HOTAIR in sepsis. Cell viability was assessed using the CCK-8 kit, while inflammation responses were measured using ELISA for IL-1β, IL-6, and TNF-α. Additionally, we employed qPCR, MeRIP, and RIP to further explore the underlying mechanisms. ResultsOur findings indicate that dexmedetomidine treatment enhanced cell viability and reduced the production of inflammatory cytokines in LPS-treated RAW264.7 cells. Furthermore, we observed that the expression of HOTAIR was increased in LPS-treated RAW264.7 cells, which was then decreased upon dexmedetomidine pre-treatment. Further investigation demonstrated that HOTAIR could counteract the beneficial effects of dexmedetomidine on cell viability and cytokine production. Interestingly, we discovered that YTHDF1 targeted HOTAIR and was upregulated in LPS-treated RAW264.7 cells, but reduced in dexmedetomidine treatment. We also found that YTHDF1 increased HOTAIR and HOTAIR m6A levels. ConclusionsCollectively, our results suggest that dexmedetomidine downregulates HOTAIR and YTHDF1 expression, which in turn inhibits the biological behavior of LPS-treated RAW264.7 cells. This finding has potential implications for the prevention and treatment of sepsis-induced kidney injury.

YTHDF1's Regulatory Involvement in Breast Cancer Prognosis, Immunity, and the ceRNA Network.

YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), an m6A reader, has a role in the development and progression of breast cancer as well as the immunological microenvironment. The networks of competing endogenous RNA in cancer have received much attention in research. In tumor gene therapy, the regulatory networks of m6A and competing endogenous RNA are increasingly emerging as a new route. We evaluated the relationship between the YTHDF1 expression, overall survival, and clinicopathology of breast cancer using TCGA, PrognoScan, and other datasets. We used Western blot to demonstrate that YTHDF1 is substantially expressed in breast cancer tissues. Furthermore, we explored YTHDF1's functions in the tumor mutational burden, microsatellite instability, and tumor microenvironment. Our findings indicate that YTHDF1 is a critical component of the m6A regulatory proteins in breast cancer and may have a particular function in the immunological microenvironment. Crucially, we investigated the relationship between YTHDF1 and the associated competitive endogenous RNA regulatory networks, innovatively creating three such networks (Dehydrogenase/Reductase 4-Antisense RNA 1-miR-378g-YTHDF1, HLA Complex Group 9-miR-378g-YTHDF1, Taurine Up-regulated 1-miR-378g-YTHDF1). Furthermore, we showed that miR-378g could inhibit the expression of YTHDF1, and that miR-378g/YTHDF1 could impact MDA-MB-231 proliferation. We speculate that YTHDF1 may serve as a biomarker for poor prognosis and differential diagnosis, impact the growth of breast cancer cells via the ceRNA network axis, and be a target for immunotherapy against breast cancer.

N6-Methyladenosine Reader YTHDF1 Promotes Stemness and Therapeutic Resistance in Hepatocellular Carcinoma by Enhancing NOTCH1 Expression.

Inhibition of YTHDF1 expression suppresses stemness of hepatocellular carcinoma cells and enhances sensitivity to targeted therapies, indicating that targeting YTHDF1 may be a promising therapeutic strategy for liver cancer.

Ferroptosis, M6A and immune checkpoint-related gene expression in the middle temporal gyrus of the Alzheimer's disease brain

Alzheimer's disease (AD) is a common genetically related cognitive disorder. Studies have shown that ferroptosis, N⁶-Methyladenosine (M6A) and immune checkpoint are related to the development of AD. However, the effects of these three gene pathways on AD progression are still unclear. Here, we used genes expressed in the middle temporal gyrus (MTG) to study the differences in ferroptosis, M6A and immune checkpoint-related gene in 97 Alzheimer's disease and 98 normal controls (NC). We then conducted correlation analysis between ferroptosis, M6A and immune checkpoint-related gene expression levels to investigate the relationship between these genes and AD. Compared to the NC, the gene expression from MTG in AD are as follows: (1) in ferroptosis related genes, the expression of CARS, CDKN1A, HSPB1, MT1G, EMC2, SAT1 and SLC1A5 was increased, while the expression of ACSL4, ATP5MC3, CSID1, CS, DPP4, GLS2 and GPX4 was decreased; (2) in M6A-related genes, the expression of HNRNPA2B1, IGF2BP2, RBM15B and YTHDC1 was increased, while the expression of FTO, YTHDC2 and YTHDF2 was decreased; (3) the expression of immune checkpoint-related genes (including CTLA4, HAVCR2 and LAG3) was increased. Further, we determined related gene pathways among these genes by conducting a literature review. By verifying the dataset, we can well verify our results and prove that our results have good robustness. We concluded of gene expression that a complete set of ferroptosis, M6A and immune checkpoint regulatory mechanisms is activated in the MTG during AD development.

YTHDF3 modulates the progression of breast cancer cells by regulating FGF2 through m6A methylation.

Breast cancer (BC) is a prevailing malignancy among women, and its inconspicuous development contributes significantly to mortality. The RNA N6-methyladenosine (m6A) modification represents an emerging mechanism for gene expression regulation, with the active involvement of the YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) in tumor progression across multiple cancer types. Nonetheless, its precise function in breast cancer necessitates further investigation. The expression of YTHDF3 in both cell lines and patient tissues was examined using Western blotting, reverse transcription quantitative PCR (RT-qPCR), and immunohistochemistry (IHC) techniques. Bioinformatics analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptome RNA sequencing (RNA-seq) data was employed to screen for the target genes of YTHDF3. The main focus of this study was to investigate the in vitro biological functions of YTHDF3. The specific binding of YTHDF3 to its target genes and its correlation with m6A methylation were studied through RNA pull-down, RNA immunoprecipitation, and co-immunoprecipitation experiments. The protein regulatory mechanisms of downstream genes of YTHDF3 were assessed using protein stability analysis. Furthermore, the biological functions of YTHDF3 and its target genes in breast cancer cells were validated through CRISPR-Cas9 technology and rescue experiments. By constructing a risk model using the TCGA database, YTHDF3 was identified as a high-risk factor among m6A methylation factors. Subsequent investigations revealed its elevated expression in various subtypes of breast cancer, accompanied by poor prognosis. MeRIP-seq analysis further revealed fibroblast growth factor 2 (FGF2) as a downstream gene of YTHDF3. Knockdown of YTHDF3 in breast cancer cells led to significant inhibition of cell self-renewal, migration, and invasion abilities in vitro. Mechanistically, YTHDF3 specifically recognized the methylated transcript of FGF2 within its coding sequence (CDS) region, leading to the inhibition of FGF2 protein degradation. Moreover, depletion of FGF2 markedly suppressed the biological functions of breast cancer cells, while reducing FGF2 expression in YTHDF3-overexpressing breast cancer cell lines substantially alleviated the malignant progression. In summary, our study elucidates the role of YTHDF3 as an oncogene in maintaining FGF2 expression in BC cells through an m6A-dependent mechanism. Additionally, we provide a potential biomarker panel for prognostic prediction in BC.

Clinical significance of the m6A methyltransferase METTL3 in peripheral blood of patients with coronary heart disease.

This study aims to explore the association of methyltransferase-like protein 3 (METTL3) expression with severity of coronary artery stenosis in patients with coronary heart disease (CHD). A total of 100 patients administrated in the Fourth Affiliated Hospital of China Medical University between October 2022 and June 2023 with primary symptoms of chest pain or tightness, or cardiac discomfort, and who underwent coronary angiography for a definitive diagnosis, were included in the study. The baseline characteristics, including TG, TC, LDL-C, HDL-C, uric acid and past history were recorded. Peripheral blood samples were collected to assess the expression levels of METTL3, YT521-B homology domains 1 (YTHDF1), YT521-B homology domains 2 (YTHDF2), and YT521-B homology domains 3 (YTHDF3) using the PCR method. Relative expression levels of METTL3 protein were determined by Western blotting. Correlation analysis were conducted to evaluate the relationship between METTL3/YTHDF1 gene expression and clinical data. Receiver operating characteristic (ROC) curve analysis was employed to assess the predictive value of METTL3 and YTHDF1 for CHD. Binary logistic regression was used to determine whether the expression of METTL3 and YTHDF1 in peripheral blood were risk factors for CHD. The study found no significant differences in baseline characteristics between CHD patients and controls, except for length of stay, Lymphocytes, Neutrophils, AST, HDL-C and modified Gensini score. The gene expression levels of METTL3 and YTHDF1 were significantly higher in CHD patients compared to controls (p < 0.05). Furthermore, METTL3 protein expression was also significantly elevated in the CHD group compared to the control group (p < 0.05). METTL3 gene expression correlated with HDL-C and Gensini score, while YTHDF1 gene expression correlated with Age, WBC, Neutrophils, RDW-CV, modified Gensini score. ROC curve analysis demonstrated an AUC of 0.692 for METTL3 in CHD, with a sensitivity of 66.7% and a specificity of 69.8% at a cut-off value of >0.052. The AUC for YTHDF1 in CHD was 0.623, with a sensitivity of 47.4% and a specificity of 74.4% at a cut-off value of >0.027. Binary logistic regression revealed that only increased METTL3 expression in peripheral blood was an independent risk factor for CHD (p < 0.05). The increased expression of METTL3 in peripheral blood may serve as a potential biomarker and predictive factor for CHD.

METTL3-mediated m6A RNA methylation was involved in aluminum-induced neurotoxicity

Aluminum (Al) exposure has been linked to the development of a variety of neurodegenerative diseases. However, whether m6A RNA methylation participated in Al-induced neurotoxicity remain to be defined. In this study, mice were administrated with aluminum-lactate at dose of 220 mg/kg. bw by gavage for 3 months. Meanwhile, the primary hippocampal neurons were isolated and treated with 0, 50, 100, 150 μM aluminum-lactate, respectively for 7 days. Al exposure caused neuronal shrinkage, decreased Nissl bodies, and increased apoptosis. In accordance, in vitro studies also showed that Al exposure led to neuronal apoptosis in a dose-dependent manner, together with the decline in m6A RNA methylation levels. Moreover, the mRNA expression of Mettl3, Mettl14, Fto, and Ythdf2 were decreased upon Al exposure. Notably, the protein expression of METTL3 was dramatically down-regulated by 42% and 35% in Al-treated mice and neurons, suggesting METTL3 might exert a crucial role in Al-induced neurotoxicity. We next established a mouse model with hippocampus-specific overexpressing of Mettl3 gene to confirm the regulatory role of RNA methylation and found that METTL3 overexpression relieved the neurological injury induced by Al. The integrated MeRIP-seq and RNA-seq analysis elucidated that 631 genes were differentially expressed at both m6A RNA methylation and mRNA expression. Notably, EGFR tyrosine kinase inhibitor resistance, Rap1 signaling pathway, protein digestion and absorption might be involved in Al-induced neurotoxicity. Moreover, VEGFA, Thbs1, and PDGFB might be the central molecules. Collectively, our findings provide the novel sight into the role of m6A RNA methylation in neurodegenerative disease induced by Al.

A Novel Pyroptosis-Based Prognostic Model Correlated with the Parainflammatory Immune Microenvironment of Pancreatic Cancer

Background. Pyroptosis has a dual function in malignant tumor progression and management. The action of pyroptosis-related genes (PRGs) in pancreatic cancer (PC), however, remains uncertain. Methods. Differential expression analyses of 57 PRGs were conducted in the TCGA TARGET GTEx dataset. The candidate genes were determined using LASSO Cox regression and random forest analyses. A risk model was developed with the TCGA dataset and validated with the ICGC dataset. Results. Three prognosis-related PRGs (BAK1, GSDMC, and IL18) were chosen to create a risk model. High-risk patients from the TCGA and ICGC cohorts had an unfavorable overall survival (all p&lt;0.05). The risk modelʼs accuracy and independent predictability were assessed by receiver operating characteristic curves and multivariate Cox regression analysis, respectively. High-risk patients possessed different molecular pathways, higher KRAS and TP53 mutations, increased expression of PD-L1, C1 immune subtype, and immunosuppressive microenvironment characterized by parainflammation compared to low-risk patients. KRAS and TP53 mutations participated in different inflammatory pathways and played different prognostic roles between the two risk groups. KRAS mutations in high-risk patients caused a more unfavorable prognosis than wild-type KRAS (p=0.016), whereas TP53 mutations in low-risk patients exhibited a poorer outcome than wild-type TP53 (p=0.009). Spearman correlation analyses revealed that the parainflammatory response in PC might be implicated in GSDMC-mediated pyroptosis via cytosolic DNA-sensing pathways under hypoxic conditions. Furthermore, the risk scores were significantly and positively related to the expression of HNRNPC, RBM15, YTHDF1, and YTHDF2, as well as sensitivity to gemcitabine, cisplatin, and erlotinib. Conclusions. This study created a novel pyroptosis-based risk model related to the parainflammatory immune microenvironment, which might help identify novel biomarkers, evaluate the tumor immune microenvironment, and develop management strategies for PC patients.

Comprehensive analysis of m6A reader YTHDF2 prognosis, immune infiltration, and related regulatory networks in hepatocellular carcinoma

BackgroundN6-Methyladenosine (m6A) RNA modification is the most prevalent internal modification pattern in eukaryotic mRNAs and plays critical roles in diverse physiological and pathological processes. However, the expression of m6A regulator YTHDF2, its prognostic value, its biological function, its correlation with tumor microenvironment (TME) immune infiltrates, and related regulatory networks in hepatocellular carcinoma (HCC) remain determined. MethodsTCGA, GTEx, and GEO databases were used to investigate the expression profile of YTHDF2 in HCC. We performed differentially expressed genes (DEGs) analysis and constructed a PPI network to explore the biological processes of YTHDF2 in HCC. Kaplan-Meier curves and Cox regression analysis were used to assess the prognostic value of YTHDF2 and then a clinical prognostic nomogram was constructed. Additionally, ssGSEA was performed to assess the correlation between YTHDF2 and immune infiltration levels. The TISIDB database was applied to explore the expression of YTHDF2 in immune and molecular subtypes of HCC. GSEA identifies the YTHDF2-related signaling pathways. Finally, we utilized miRNet and starBase database to construct regulatory networks for HCC based on lncRNA-miRNA and miRNA-YTHDF2 interactions. ResultsYTHDF2 was significantly upregulated in HCC tumor tissues compared with the adjacent normal tissues. HCC patients in the high YTHDF2 expression group had poorer survival. Multivariate Cox analysis suggested that YTHDF2 may be a new independent prognostic indicator for HCC patients, with the prognostic nomogram exhibiting satisfactory results. YTHDF2 expression was significantly correlated with TME immune cell-infiltrating characteristics. Strong correlations were also shown in immune subtypes, molecular subtypes and immune checkpoints. Further analysis revealed that the combination of YTHDF2 expression and immune cell score was considerably associated with survival outcome in HCC patients. GESA analysis demonstrated that high YTHDF2 expression is associated with multiple biological processes and oncogenic pathways. Moreover, 14 possible regulatory networks were constructed, which are associated with HCC progression. ConclusionOur findings revealed that YTHDF2 may serve as a promising prognostic biomarker for HCC and may regulate the tumor immune microenvironment to provide effective therapeutic strategies.

YTHDF1 enhances stemness and chemoresistance in triple-negative breast cancer cells by upregulating SIAH2.

Triple-negative breast cancer (TNBC) is the most lethal and aggressive subtype of breast cancer, and chemoresistance is the major determinant of TNBC treatment failure. This study explores the molecular mechanism of TNBC chemoresistance.The Cancer Genome Atlas, breast cancer integrative platform, and GEPIA databases were used to analyze the expression and correlation of YTHDF1 and seven in absentia homology 2 (SIAH2) in breast cancer. Knockdown of YTHDF1 and SIAH2, or overexpression of SIAH2 in vitro and in vivo, was conducted to evaluate the impact of changes in YTHDF1 and SIAH2 expression on TNBC cell proliferation, apoptosis, stemness, drug resistance, and Hippo pathway gene expression.YTHDF1 and SIAH2 were highly expressed in breast cancer patients and TNBC cells. Knockdown of YTHDF1 and SIAH2 significantly inhibited proliferation and stemness and promoted apoptosis and chemosensitivity of TNBC cells. Mechanistically, the knockdown of YTHDF1 inhibited the expression of SIAH2, thereby downregulating the Hippo pathway, which inhibited proliferation and stemness and promoted apoptosis and chemosensitivity of TNBC cells.The current findings revealed the regulatory mechanism of YTHDF1 in TNBC and clarified the role of the YTHDF1/SIAH2 axis in TNBC drug resistance and stemness. This could provide new insights into the vital role of targeting YTHDF1/SIAH2 to suppress drug resistance and stemness in TNBC cells.

Upregulation of eIF2α by m6A modification accelerates the proliferation of pulmonary artery smooth muscle cells in MCT-induced pulmonary arterial hypertension rats.

Pulmonary arterial hypertension (PAH) is a malignant cardiovascular disease. Eukaryotic initiation factor 2α (eIF2α) plays an important role in the proliferation of pulmonary artery smooth muscle cells (PASMCs) in hypoxia-induced pulmonary hypertension (HPH) rats. However, the regulatory mechanism of eIF2α remains poorly understood in PAH rats. Here, we discover eIF2α is markedly upregulated in monocrotaline (MCT)-induced PAH rats, eIF2α can be upregulated by mRNA methylation, and upregulated eIF2α can promote PASMC proliferation in MCT-PAH rats. GSK2606414, eIF2α inhibitor, can downregulate the expression of eIF2α and alleviate PASMC proliferation in MCT-PAH rats. And we further discover the mRNA of eIF2α has a common sequence with N 6-methyladenosine (m6A) modification by bioinformatics analysis, and the expression of METTL3, WTAP, and YTHDF1 is upregulated in MCT-PAH rats. These findings suggest a potentially novel mechanism by which eIF2α is upregulated by m6A modification in MCT-PAH rats, which is involved in the pathogenesis of PAH.

YTHDF2 Promotes Cardiac Ferroptosis via Degradation of SLC7A11 in Cardiac Ischemia-Reperfusion Injury.

Myocardial ischemia-reperfusion (I/R) injury facilitates cardiomyocyte death and endangers human health. N6-methyladenosine (m6A) methylation plays a critical role in cardiovascular diseases. The m6A reader YTHDF2 identifies m6A-modified RNA and promotes target RNA degradation. Hence, we hypothesized that YTHDF2 affects I/R injury by regulating RNA stability. Both mRNA and protein levels of YTHDF2 were upregulated in I/R mice and hypoxia-reoxygenation (H/R)-induced cardiomyocytes. Silencing of endogenous YTHDF2 abrogated cardiac dysfunction and lowered the infarct size in I/R mice, and forced expression of YTHDF2 aggravated these adverse pathological processes. Consistently, the protective effect of silencing YTHDF2 occurred in cardiomyocytes exposed to H/R and erastin. Furthermore, RNA-seq and RIP revealed that YTHDF2 recognized the m6A modification sites of the ferroptosis-related gene SLC7A11 mRNA to promote its degradation both in vivo and in vitro. Inhibition of SLC7A11 impaired cardiac function, increased infarct size, and the release of LDH in I/R mice after silencing YTHDF2. The beneficial effects of si-YTHDF2 on H/R injury were reversed by co-transfection with si-SLC7A11, which substantially exacerbated ferroptosis and the production of ROS. The cardioprotective effects of silencing YTHDF2 are accomplished by increasing SLC7A11 stability and expression and reducing ferroptosis, providing novel potential therapeutic targets for treating ischemic cardiac diseases.

Bioinformatics analysis identifies potential m6A hub genes in the pathogenesis of intracerebral hemorrhage

BackgroundIntracerebral hemorrhage (ICH) is a type of stroke associated with a high rate of disability and mortality. The role of N6-methyladenosine (m6A) in ICH remains unclear. MethodsScreening of m6A DEGs by differentially expressed genes (DEGs) analysis. m6A hub genes in ICH were identified by protein-protein interaction (PPI) networks. Pearson correlation tests were used to explore the relationship between m6A hub genes and DNA methylation. m6A hub genes were examined by ROC curves for their ability to predict ICH occurrence. Immune cell infiltration and m6A hub gene correlation in ICH were analysed using the CIBERSORT algorithm. Construction of ceRNA networks and enrichment analysis by GO/KEGG. ResultsA total of 12 m6A regulatory enzymes were differentially expressed after ICH. the PPI network screened three m6A hub genes, including YTHDF2, FTO and HNRNPA2B1. A high expression of YTHDF2 was associated with DNA hypomethylation after ICH and could better predict the development of ICH. yTHDF2 was associated with high infiltration of M1 macrophages after ICH. A ceRNA network was constructed based on the m6A central gene with target genes enriched in transcriptional regulation and the LKB1 signalling pathway. ConclusionM6A modifications are involved in the progression of ICH. YTHDF2, an m6A key gene, may regulate ICH progression by promoting infiltration of M1 macrophages or through the ceRNA network.

Targeting YTHDF2 inhibits tumorigenesis of diffuse large B-cell lymphoma through ACER2-mediated ceramide catabolism

IntroductionEpigenetic alterations play crucial roles in diffuse large B-cell lymphoma (DLBCL). Disturbances in lipid metabolism contribute to tumor progression. However, studies in epigenetics, especially its critical regulator YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), on lipid metabolism regulation in DLBCL are unidentified. ObjectivesElucidate the prognostic value and biological functions of YTHDF2 in DLBCL and illuminate the underlying epigenetic regulation mechanism of lipid metabolism by YTHDF2 in DLBCL development. MethodsThe expression and clinical value of YTHDF2 in DLBCL were performed in public databases and clinical specimens. The biological functions of YTHDF2 in DLBCL were determined in vivo and in vitro through overexpression and CRISPR/Cas9-mediated knockout of YTHDF2. RNA sequencing, lipidomics, methylated RNA immunoprecipitation sequencing, RNA immunoprecipitation-qPCR, luciferase activity assay, and RNA stability experiments were used to explore the potential mechanism by which YTHDF2 contributed to DLBCL progression. ResultsYTHDF2 was highly expressed in DLBCL, and related to poor prognosis. YTHDF2 overexpression exerted a tumor-promoting effect in DLBCL, and knockdown of YTHDF2 restricted DLBCL cell proliferation, arrested cell cycle in the G2/M phase, facilitated apoptosis, and enhanced drug sensitivity to ibrutinib and venetoclax. In addition, YTHDF2 knockout drastically suppressed tumor growth in xenograft DLBCL models. Furthermore, a regulatory role of YTHDF2 in ceramide metabolism was identified in DLBCL cells. Exogenous ceramide effectively inhibited the malignant phenotype of DLBCL cells in vitro. The binding of YTHDF2 to m6A sites on alkaline ceramidase 2 (ACER2) mRNA promoted its stability and expression. Enhanced ACER2 expression hydrolyzed ceramides, disrupting the balance between ceramide and sphingosine-1-phosphate (S1P), activating the ERK and PI3K/AKT pathways, and leading to DLBCL tumorigenesis. ConclusionThis study demonstrated that YTHDF2 contributed to the progression of DLBCL by regulating ACER2-mediated ceramide metabolism in an m6A-dependent manner, providing novel insights into targeted therapies.

Hypothalamic expression analysis of m6A RNA methylation associated genes suggests a potential role of epitranscriptomics in sexual maturation of Atlantic salmon

Better understanding the molecular processes contributing to variation in maturation timing is important for Atlantic salmon (Salmo salar) aquaculture, as early maturation causes considerable financial losses. The m6A RNA methylation is a highly conserved and dynamically reversible mechanism controlling gene expression in a myriad of biological processes. The role of m6A methylation in sexual maturation, however, has remained largely unexplored and has never been studied in Atlantic salmon. While the maturation process is known to be affected by many genetic and environmental factors, the molecular mechanisms causing variation in the timing of maturation are still poorly understood. Hence, investigation of whether a widespread mechanism like m6A methylation could be involved in controlling of the maturation timing is warranted. In Atlantic salmon, two genes, also associated with age at maturity in humans, vgll3 and six6, have been shown to play an important role in maturation timing. In this study, we investigated the expression of 16 genes involved in the regulation of m6A RNA methylation in the hypothalamus of Atlantic salmon with different homozygous combinations of late (L) and early (E) alleles for vgll3 and six6 genes. We found differential expression of ythdf2.2, an m6A reader promoting mRNA degradation, with higher expression in six6*LL compared to other genotypes as well as in immature compared to mature males. In addition, we found that the expression levels of genes coding for an eraser, alkbh5, and for a reader, ythdf1, were higher in the hypothalamus of females than in males across all the different genotypes studied. However the total m6A levels between the whole hypothalamus of males and females were similar. Our results indicate a potential role of the m6A methylation process in sexual maturation of Atlantic salmon, and therefore, provide the first evidence for such regulatory mechanism in the hypothalamus of any vertebrate.

YTHDF1 shapes “cold” tumor and inhibits CD8+ T cells infiltration and function in breast cancer

While YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) was recognized as a crucial contributor in the development and immune-related regulation of various types of tumors, its function in the immune response of breast cancer has largely remained uninvestigated. Through analysis of public databases, we found YTHDF1 as a highly expressed gene in breast cancers and confirmed this finding in breast cancer cells and clinical specimens from our center. Subsequently, we examined the link between YTHDF1 expression and immune cells and molecules by utilizing immune-related public databases and algorithm. We further validated our findings through cellular and animal experiments, as well as RNA sequencing. YTHDF1 was found highly expressed in tumor tissues of breast cancer, which negatively correlated with patient survival. The downregulation of YTHDF1 promoted the expression of pro-inflammatory markers and improved the anti-cancer ability of immune cells in breast cancer. RNA sequencing analysis revealed that YTHDF1 knockdown resulted in enrichment of differential genes in signal transduction pathways. Additionally, in vitro experiments showed that immune cells had higher cytotoxicity against breast cancer cells with decreased YTHDF1 expression. Moreover, in vivo studies indicated that YTHDF1 promoted breast cancer growth while inhibiting CD8+ T cell infiltration and function. Our study demonstrates that YTHDF1 plays a crucial role in establishing a “cold” tumor microenvironment in breast cancer by inhibiting the release of pro-inflammatory cytokines from cancer cells. As a result, the infiltration and functional differentiation of anti-tumor CD8+ T cells are hindered, ultimately resulting in the immune evasion of breast cancer.

Curdione induces ferroptosis mediated by m6A methylation via METTL14 and YTHDF2 in colorectal cancer

BackgroundCurdione is a sesquiterpene isolated from Curcumae Rhizoma that possesses high biological activity and extensive pharmacological effects. As a traditional Chinese medicine, Curcumae Rhizoma can inhibit the development of many types of cancer, especially colorectal cancer. However, the anti-colorectal mechanism of its monomer curdione remains unclear.MethodsColorectal cancer (CRC) cells were treated with curdione at doses of 12.5 μM, 25 μM, and 50 μM, and then the cells’ activity was measured with methyl thiazolyl tetrazolium (MTT). Nude mice were administered different doses of curdione subcutaneously and oxaliplatin by tail vein injection, and then hematoxylin–eosin (HE) staining was adopted to examine tumor histology. Moreover, flow cytometry was applied to detect reactive oxygen species in cells and tissues. Kits were employed to detect the levels of iron ions, malondialdehyde, lipid hydroperoxide, and glutathione. Polymerase chain reaction (PCR) and Western blotting were adopted to detect ferroptosis and m6A modification-related factors. A methylation spot hybridization assay was performed to measure changes in overall methylation. SLC7A11 and HOXA13 were measured by MeRIP-qPCR. The shRNA-METTL14 plasmid was constructed to verify the inhibitory effect of curdione on CRC.ResultsA dose-dependent decrease in activity was observed in curdione-treated cells. Curdione increased the accumulation of reactive oxygen species in CRC cells and tumor tissues, greatly enhanced the levels of malondialdehyde, lipid hydroperoxide and Fe2+, and lowered the activity of glutathione. According to the qPCR and Western blot results, curdione promoted the expression of METTL14 and YTHDF2 in CRC cells and tissues, respectively, and decreased the expression of SLC7A11, SLC3A2, HOXA13, and glutathione peroxidase 4. Additionally, in animal experiments, the curdione-treated group showed severe necrosis of tumor cells, as displayed by HE staining. Furthermore, compared with the control group, levels of m6A modifying factors (namely, SLC7A11 and HOXA13) were increased in the tissues after drug intervention. METTL14 knockdown was followed by an increase in CRC cell activity and glutathione levels. However, the levels of reactive oxygen species, malondialdehyde, and iron ions decreased. The expression levels of SLC7A11, SLC3A2, HOXA13, and GPX4 were all increased after METTL14 knockdown.ConclusionThe results suggest that curdione induces ferroptosis in CRC by virtue of m6A methylation.