A Unified Model for the Function of YTHDF Proteins in Regulating m6A-Modified mRNA.

N6-methyladenosine (m6A) is the most abundant mRNA nucleotide modification and regulates critical aspects of cellular physiology and differentiation. m6A is thought to mediate its effects through a complex network of interactions between different m6A sites and three functionally distinct cytoplasmic YTHDF m6A-binding proteins (DF1, DF2, and DF3). In contrast to the prevailing model, we show that DF proteins bind the same m6A-modified mRNAs rather than different mRNAs. Furthermore, we find that DF proteins do not induce translation in HeLa cells. Instead, the DF paralogs act redundantly to mediate mRNA degradation and cellular differentiation. The ability of DF proteins to regulate stability and differentiation becomes evident only when all three DF paralogs are depleted simultaneously. Our study reveals a unified model of m6A function in which all m6A-modified mRNAs are subjected to the combined action of YTHDF proteins in proportion to the number of m6A sites.

FMRP phosphorylation modulates neuronal translation through YTHDF1.

RNA-binding proteins (RBPs) control messenger RNA fate in neurons. Here, we report a mechanism that the stimuli-induced neuronal translation is mediated by phosphorylation of a YTHDF1-binding protein FMRP. Mechanistically, YTHDF1 can condense with ribosomal proteins to promote the translation of its mRNA targets. FMRP regulates this process by sequestering YTHDF1 away from the ribosome; upon neuronal stimulation, FMRP becomes phosphorylated and releases YTHDF1 for translation upregulation. We show that a new small molecule inhibitor of YTHDF1 can reverse fragile X syndrome (FXS) developmental defects associated with FMRP deficiency in an organoid model. Our study thus reveals that FMRP and its phosphorylation are important regulators of activity-dependent translation during neuronal development and stimulation and identifies YTHDF1 as a potential therapeutic target for FXS in which developmental defects caused by FMRP depletion could be reversed through YTHDF1 inhibition.

Hidden codes in mRNA: Control of gene expression by m6A.

Information in mRNA has largely been thought to be confined to its nucleotide sequence. However, the advent of mapping techniques to detect modified nucleotides has revealed that mRNA contains additional information in the form of chemical modifications. The most abundant modified nucleotide is N6-methyladenosine (m6A), a methyl modification of adenosine. Although early studies viewed m6A as a dynamic and tissue-specific modification, it is now clear that the mRNAs that contain m6A and the location of m6A in those transcripts are largely universal and are influenced by gene architecture, i.e., the size and location of exons and introns. m6A can affect nuclear processes such as splicing and epigenetic regulation, but the major effect of m6A on mRNAs is to promote degradation in the cytoplasm. m6A marks a functionally related cohort of mRNAs linked to certain biological processes, including cell differentiation and cell fate determination. m6A is also enriched in other cohorts of mRNAs and can therefore affect their respective cellular processes and pathways. Future work will focus on understanding how the m6A pathway is regulated to achieve control of m6A-containing mRNAs.

Epitranscriptic regulation of HRAS by N6-methyladenosine drives tumor progression.

Overexpression of Ras, in addition to the oncogenic mutations, occurs in various human cancers. However, the mechanisms for epitranscriptic regulation of RAS in tumorigenesis remain unclear. Here, we report that the widespread N6-methyladenosine (m6A) modification of HRAS, but not KRAS and NRAS, is higher in cancer tissues compared with the adjacent tissues, which results in the increased expression of H-Ras protein, thus promoting cancer cell proliferation and metastasis. Mechanistically, three m6A modification sites of HRAS 3' UTR, which is regulated by FTO and bound by YTHDF1, but not YTHDF2 nor YTHDF3, promote its protein expression by the enhanced translational elongation. In addition, targeting HRAS m6A modification decreases cancer proliferation and metastasis. Clinically, up-regulated H-Ras expression correlates with down-regulated FTO and up-regulated YTHDF1 expression in various cancers. Collectively, our study reveals a linking between specific m6A modification sites of HRAS and tumor progression, which provides a new strategy to target oncogenic Ras signaling.

YTHDF1 is pivotal for maintenance of cardiac homeostasis.

The YTH-domain family (YTHDF) of RNA binding proteins can control gene expression at the post-transcriptional level by regulating mRNAs with N6-methyladenosine (m6A) modifications. Despite the established importance of m6A in the heart, the cardiac role of specific m6A-binding proteins remains unclear. Here, we characterized the function of YTHDF1 in cardiomyocytes using a newly generated cardiac-restricted mouse model. Deletion of YTHDF1 in adult cardiomyocytes led to hypertrophy, fibrosis, and dysfunction. Using mass spectrometry, we identified the necessity of YTHDF1 for the expression of cardiomyocyte membrane raft proteins. Specifically, YTHDF1 bound to m6A-modified Caveolin 1 (Cav1) mRNA and favored its translation. We further demonstrated that YTHDF1 regulates downstream ERK signaling. Altogether, our findings highlight a novel role for YTHDF1 as a post-transcriptional regulator of caveolar proteins which is necessary for the maintenance of cardiac function.

The ubiquitin ligase STUB1 suppresses tumorigenesis of renal cell carcinomas through regulating YTHDF1 stability.

STIP1 homology and U-box protein 1 (STUB1), a crucial member of the RING family E3 ubiquitin ligase, serves dual roles as an oncogene and a tumor suppressor in various human cancers. However, the role and mechanism of STUB1 in clear cell renal cell carcinoma (ccRCC) remain poorly defined. Here, we identified YTHDF1 as a novel STUB1 interaction partner using affinity purification mass spectrometry (AP-MS). Furthermore, we revealed that STUB1 promotes the ubiquitination and degradation of YTHDF1. Consequently, STUB1 depletion leads to YTHDF1 up-regulation in renal cancer cells. Functionally, STUB1 depletion promoted migration and invasion of ccRCC cells in a YTHDF1-dependent manner. Additionally, depletion of STUB1 also increased the tumorigenic potential of ccRCC in a xenograft model. Importantly, STUB1 expression is down-regulated in ccRCC tissues, and its low expression level correlates with advanced tumor stage and poor overall survival in ccRCC patients. Taken together, these findings reveal that STUB1 inhibits the tumorigenicity of ccRCC by regulating YTHDF1 stability.

YTHDF1 promotes gallbladder cancer progression via post-transcriptional regulation of the m6A/UHRF1 axis.

Gallbladder cancer is a rare but fatal malignancy. However, the mechanisms underlying gallbladder carcinogenesis and its progression are poorly understood. The function of m6A modification and its regulators was still unclear for gallbladder cancer. The current study seeks to investigate the function of YTH m6A RNA-binding protein 1 (YTHDF1) in gallbladder cancer. Transcriptomic analysis and immunochemical staining of YTHDF1 in gallbladder cancer tissues revealed its upregulation compared to paracancerous tissues. Moreover, YTHDF1 promotes the proliferation assays, Transwell migration assays, and Transwell invasion assays of gallbladder cancer cells in vitro. And it also increased tumour growth in xenograft mouse model and metastases in tail vein injection model in vivo. In vitro, UHRF1 knockdown partly reversed the effects of YTHDF1 overexpression. Mechanistically, dual-luciferase assays proved that YTHDF1 promotes UHRF1 expression via direct binding to the mRNA 3'-UTR in a m6A-dependent manner. Overexpression of YTHDF1 enhanced UHRF1 mRNA stability, as demonstrated by mRNA stability assays, and Co-IP studies confirmed a direct interaction between YTHDF1 and PABPC1. Collectively, these findings provide new insights into the progression of gallbladder cancer as well as a novel post-transcriptional mechanism of YTHDF1 via stabilizing target mRNA.

O-GlcNAcylation promotes the cytosolic localization of the m6A reader YTHDF1 and colorectal cancer tumorigenesis.

O-linked GlcNAc (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalk with phosphorylation and ubiquitination to orchestrate various biological processes. The mechanisms underlying the involvement of O-GlcNAc modifications in N6-methyladenosine (m6A) regulation are not fully characterized. Herein, we show that O-GlcNAc modifies the m6A mRNA reader YTH domain family 1 (YTHDF1) and fine-tunes its nuclear translocation by the exportin protein Crm1. First, we present evidence that YTHDF1 interacts with the sole O-GlcNAc transferase (OGT). Second, we verified Ser196/Ser197/Ser198 as the YTHDF1 O-GlcNAcylation sites, as described in numerous chemoproteomic studies. Then we constructed the O-GlcNAc-deficient YTHDF1-S196A/S197F/S198A (AFA) mutant, which significantly attenuated O-GlcNAc signals. Moreover, we revealed that YTHDF1 is a nucleocytoplasmic protein, whose nuclear export is mediated by Crm1. Furthermore, O-GlcNAcylation increases the cytosolic portion of YTHDF1 by enhancing binding with Crm1, thus upregulating downstream target (e.g. c-Myc) expression. Molecular dynamics simulations suggest that O-GlcNAcylation at S197 promotes the binding between the nuclear export signal motif and Crm1 through increasing hydrogen bonding. Mouse xenograft assays further demonstrate that YTHDF1-AFA mutants decreased the colon cancer mass and size via decreasing c-Myc expression. In sum, we found that YTHDF1 is a nucleocytoplasmic protein, whose cytosolic localization is dependent on O-GlcNAc modification. We propose that the OGT-YTHDF1-c-Myc axis underlies colorectal cancer tumorigenesis.

YTHDF1 facilitates PRC1-mediated H2AK119ub in human ES cells.

Polycomb repressive complexes (PRCs) play critical roles in cell fate decisions during normal development as well as disease progression through mediating histone modifications such as H3K27me3 and H2AK119ub. How exactly PRCs recruited to chromatin remains to be fully illuminated. Here, we report that YTHDF1, the N6-methyladenine (m6 A) RNA reader that was previously known to be mainly cytoplasmic, associates with RNF2, a PRC1 protein that mediates H2AK119ub in human embryonic stem cells (hESCs). A portion of YTHDF1 localizes in the nuclei and associates with RNF2/H2AK119ub on a subset of gene loci related to neural development functions. Knock-down YTHDF1 attenuates H2AK119ub modification on these genes and promotes neural differentiation in hESCs. Our findings provide a noncanonical mechanism that YTHDF1 participates in PRC1 functions in hESCs.

N6-methyladenosine regulator YTHDF1 represses the CD8 + T cell-mediated antitumor immunity and ferroptosis in prostate cancer via m6A/PD-L1 manner.

Increasing data and literature have illustrated that tumor immune escape represents a major source of tumor formation and recrudesce. Besides, novel findings also indicate that RNA N6-methyladenosine (m6A) participates in the human cancer immune escape. Here, our study investigated the functions of m6A reader YTHDF1 in prostate cancer (PCa) immune response and explored the functional mechanism. Results reported that YTHDF1 up-regulated in PCa samples and was closely correlated to poor clinical prognosis. Functionally, YTHDF1 inhibited the killing activity of CD8 + T cells to PCa cells, and moreover mitigated the ferroptosis. Mechanistically, PD-L1 acted as the target of YTHDF1, and YTHDF1 upregulated the transcriptional activity of PD-L1 mRNA. Collectively, YTHDF1 promoted functional PD-L1 partially through enhancing its transcriptional stability, which was necessary for PCa cells to evade effector T cell cytotoxicity and CD8 + T cells mediated ferroptosis. In conclusion, these findings indicate that YTHDF1 represses the CD8 + T cell-mediated antitumor immunity and ferroptosis in PCa via m6A-PD-L1 manner, which may provide novel insight for PCa immunotherapy.

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.

DUSP5 regulated by YTHDF1-mediated m6A modification promotes epithelial-mesenchymal transition and EGFR-TKI resistance via the TGF-ß/Smad signaling pathway in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) patients have a dismal survival rate because of cancer metastasis and drug resistance. The study aims to identify the genes that concurrently modulate EMT, metastasis and EGFR-TKI resistance, and to investigate the underlying regulatory mechanisms. METHODS: Cox regression and Kaplan-Meier analyses were applied to identify prognostic oncogenes in LUAD. Gene set enrichment analysis (GSEA) was used to indicate the biological functions of the gene. Wound-healing and Transwell assays were used to detect migratory and invasive ability. EGFR-TKI sensitivity was evaluated by assessing the proliferation, clonogenic survival and metastatic capability of cancer cells with treatment with gefitinib. Methylated RNA immunoprecipitation (MeRIP) and RNA immunoprecipitation (RIP) analyses established the level of m6A modification present on the target gene and the protein's capability to interact with RNA, respectively. Single-sample gene set enrichment (ssGSEA) algorithm used to investigate levels of immune cell infiltration. RESULTS: Our study identified dual-specificity phosphatase 5 (DUSP5) as a novel and powerful predictor of adverse outcomes for LUAD by using public datasets. Functional enrichment analysis found that DUSP5 was positively enriched in EMT and transforming growth factor-beta (TGF-ß) signaling pathway, a prevailing pathway involved in the induction of EMT. As expected, DUSP5 knockdown suppressed EMT via inhibiting the canonical TGF-ß/Smad signaling pathway in in vitro experiments. Consistently, knockdown of DUSP5 was first found to inhibit migratory ability and invasiveness of LUAD cells in in vitro and prevent lung metastasis in in vivo. DUSP5 knockdown re-sensitized gefitinib-resistant LUAD cells to gefitinib, accompanying reversion of EMT progress. In LUAD tissue samples, we found 14 cytosine-phosphate-guanine (CpG) sites of DUSP5 that were negatively associated with DUSP5 gene expression. Importantly, 5'Azacytidine (AZA), an FDA-approved DNA methyltransferase inhibitor, restored DUSP5 expression. Moreover, RIP experiments confirmed that YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), a m6A reader protein, could bind DUSP5 mRNA. YTHDF1 promoted DUSP5 expression and the malignant phenotype of LUAD cells. In addition, the DUSP5-derived genomic model revealed the two clusters with distinguishable immune features and tumor mutational burden (TMB). CONCLUSIONS: Briefly, our study discovered DUSP5 which was regulated by epigenetic modification, might be a potential therapeutic target, especially in LUAD patients with acquired EGFR-TKI resistance.

YTHDF1's grip on CRC vasculature: insights into LINC01106 and miR-449b-5p-VEGFA axis.

BACKGROUND: Investigating the unexplored territory of lncRNA m6A modification in colorectal cancer (CRC) vasculature, this study focuses on LINC01106 and YTHDF1. METHODS: Clinical assessments reveal upregulated LINC01106 promoting vascular generation via the miR-449b-5p-VEGFA pathway. RESULTS: YTHDF1, elevated in CRC tissues, emerges as an adverse prognostic factor. Functional experiments showcase YTHDF1's inhibitory effects on CRC cell dynamics. Mechanistically, Me-CLIP identifies m6A-modified LINC01106, validated as a YTHDF1 target through Me-RIP. CONCLUSIONS: This study sheds light on the YTHDF1-mediated m6A modification of LINC01106, presenting it as a key player in suppressing CRC vascular generation.

KIAA1429 increases FOXM1 expression through YTHDF1-mediated m6A modification to promote aerobic glycolysis and tumorigenesis in multiple myeloma.

OBJECTIVE: Multiple myeloma (MM) is a deadly plasma cell malignancy with elusive pathogenesis. N6-methyladenosine (m6A) is critically engaged in hematological malignancies. The function of KIAA1429, the largest component of methyltransferases, is unknown. This study delved into the mechanism of KIAA1429 in MM, hoping to offer novel targets for MM therapy. METHODS: Bone marrow samples were attained from 55 MM patients and 15 controls. KIAA1429, YTHDF1, and FOXM1 mRNA levels were detected and their correlation was analyzed. Cell viability, proliferation, cell cycle, and apoptosis were testified. Glycolysis-enhancing genes (HK2, ENO1, and LDHA), lactate production, and glucose uptake were evaluated. The interaction between FOXM1 mRNA and YTHDF1, m6A-modified FOXM1 level, and FOXM1 stability were assayed. A transplantation tumor model was built to confirm the mechanism of KIAA1429. RESULTS: KIAA1429 was at high levels in MM patients and MM cells and linked to poor prognoses. KIAA1429 knockdown restrained MM cell viability, and proliferation, arrested G0/G1 phase, and increased apoptosis. KIAA1429 mRNA in plasma cells from MM patients was positively linked with to glycolysis-enhancing genes. The levels of glycolysis-enhancing genes, glucose uptake, and lactate production were repressed after KIAA1429 knockdown, along with reduced FOXM1 levels and stability. YTHDF1 recognized KIAA1429-methylated FOXM1 mRNA and raised FOXM1 stability. Knockdown of YTHDF1 curbed aerobic glycolysis and malignant behaviors in MM cells, which was nullified by FOXM1 overexpression. KIAA1429 knockdown also inhibited tumor growth in animal experiments. CONCLUSION: KIAA1429 knockdown reduces FOXM1 expression through YTHDF1-mediated m6A modification, thus inhibiting MM aerobic glycolysis and tumorigenesis.

Unraveling the molecular complexity: Wtap/Ythdf1 and Lcn2 in novel traumatic brain injury secondary injury mechanisms.

The primary aim of this research was to explore the functions of Wtap and Ythdf1 in regulating neuronal Lipocalin-2 (Lcn2) through m6A modification in traumatic brain injury (TBI). By employing transcriptome sequencing and enrichment analysis, we identified the Wtap/Ythdf1-mediated Lcn2 m6A modification pathway as crucial in TBI. In our in vitro experiments using primary cortical neurons, knockout of Wtap and Ythdf1 led to the inhibition of Lcn2 m6A modification, resulting in reduced neuronal death and inflammation. Furthermore, overexpression of Lcn2 in cortical neurons induced the activation of reactive astrocytes and M1-like microglial cells, causing neuronal apoptosis. In vivo experiments confirmed the activation of reactive astrocytes and microglial cells in TBI and importantly demonstrated that Wtap knockdown improved neuroinflammation and functional impairment. These findings underscore the significance of Wtap/Ythdf1-mediated Lcn2 regulation in TBI secondary injury and suggest potential therapeutic implications for combating TBI-induced neuroinflammation and neuronal damage.

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.

Infralimbic YTHDF1 is necessary for the beneficial effects of acute mild exercise on auditory fear extinction retention.

Exposure therapy is the most effective approach of behavioral therapy for anxiety and post-traumatic stress disorder (PTSD). But fear is easy to reappear even after successful extinction. So, identifying novel strategies for augmenting exposure therapy is rather important. It was reported that exercise had beneficial effects on cognitive and memory deficits. However, whether exercise could affect fear memory, especially for fear extinction remained elusive. Here, our results showed that exposure to acute mild exercise 1 or 2 h before extinction training can augment recent fear extinction retention and 2 h for the remote fear extinction retention. These beneficial effects could be attributed to increased YTHDF1 expression in medial prefrontal cortex (mPFC). Furthermore, by using an AAV-shRNA-based approach to silence YTHDF1 expression via stereotactic injection in prelimbic cortex (PL) or infralimbic cortex (IL), respectively, we demonstrated that silence YTHDF1 in IL, but not in PL, blunted augmentation of exposure therapy induced by acute mild exercise and accompanied with decreased NR2B and GluR1 expression. Moreover, YTHDF1 modulated dendritic spines remodeling of pyramidal neuron in IL. Collectively, our findings suggested that acute mild exercise acted as an effective strategy in augmenting exposure therapy with possible implications for understanding new treatment underlying PTSD.

Photosensitive and dual-targeted chromium nanoparticle delivering small interfering RNA YTHDF1 for molecular-targeted immunotherapy in liver cancer.

Tumor-associated macrophages (TAMs) are a promising target for cancer immunotherapy, but delivering therapeutic agents to TAMs within the tumor microenvironment (TME) is challenging. In this study, a photosensitive, dual-targeting nanoparticle system (M.RGD@Cr-CTS-siYTHDF1 NPs) was developed. The structure includes a shell of DSPE-modified RGD peptides targeting integrin receptors on tumor cells and carboxymethyl mannose targeting CD206 receptors on macrophages, with a core of chitosan adsorbing m6A reading protein YTHDF1 siRNA and chromium nanoparticles (Cr NPs). The approach is specifically designed to target TAM and cancer cells, utilizing the photothermal effect of Cr NPs to disrupt the TME and deliver siYTHDF1 to TAM. In experiments with tumor-bearing mice, M.RGD@Cr-CTS-siYTHDF1 NPs, when exposed to laser irradiation, effectively killed tumor cells, disrupted the TME, delivered siYTHDF1 to TAMs, silenced the YTHDF1 gene, and shifted the STAT3-STAT1 equilibrium by reducing STAT3 and enhancing STAT1 expression. This reprogramming of TAMs towards an anti-tumor phenotype led to a pro-immunogenic TME state. The strategy also suppressed immunosuppressive IL-10 production, increased expression of immunostimulatory factors (IL-12 and IFN-γ), boosted CD8 + T cell infiltration and M1-type TAMs, and reduced Tregs and M2-type TAMs within the TME. In conclusion, the dual-targeting M.RGD@Cr-CTS-siYTHDF1 NPs, integrating dual-targeting capabilities with photothermal therapy (PTT) and RNA interference, offer a promising approach for molecular targeted cancer immunotherapy with potential for clinical application.

m6A-YTHDF1 Mediated Regulation of GRIN2D in Bladder Cancer Progression and Aerobic Glycolysis.

The modification of N6-methyladenosine (m6A), primarily orchestrated by the reader protein YTHDF1, is a pivotal element in the post-transcriptional regulation of genes. While its role in various biological processes is well-documented, the specific impact of m6A-YTHDF1 on the regulation of GRIN2D, a gene implicated in cancer biology, particularly in the context of bladder cancer, is not thoroughly understood. Utilizing a series of bioinformatics analyses and experimental approaches, including cell culture, transfection, RT-qPCR, and western blotting, we investigated the m6A modification landscape in bladder cancer cells. The relationship between m6A-YTHDF1 and GRIN2D expression was examined, followed by functional assays to assess their roles in cancer progression and glycolytic activity. Our analysis identified a significant upregulation of m6A modification in bladder cancer tissues. YTHDF1 was found to regulate GRIN2D expression positively. Functionally, GRIN2D was implicated in promoting bladder cancer cell proliferation and enhancing aerobic glycolysis. Inhibition of the m6A-YTHDF1-GRIN2D axis resulted in the suppression of cancer progression and metabolic alterations. Through this research, we have elucidated the significant influence of the m6A-YTHDF1 axis on the modulation of GRIN2D expression, which in turn markedly impacts the progression of bladder cancer and its metabolic pathways, particularly aerobic glycolysis. Our findings uncover critical molecular dynamics within bladder cancer cells, offering a deeper understanding of its pathophysiology. Furthermore, the insights gained from this study underscore the potential of targeting the m6A-YTHDF1-GRIN2D pathway for the development of innovative therapeutic strategies in the treatment of bladder cancer.

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.