BACKGROUND: Pancreatic cancer (PC) is one of the most malignant cancers with highly aggressiveness and poor prognosis. N6-methyladenosine (m6A) have been indicated to be involved in PC development. Glucan Branching Enzyme 1 (GBE1) is mainly involved in cell glycogen metabolism. However, the function of GBE1 and Whether GBE1 occurs m6A modification in PC progression remains to be illustrated.
METHODS: The clinical prognosis of GBE1 was analyzed through online platform. The expression of GBE1 was obtained from online platform and then verified in normal and PC cell lines. Lentivirus was used to generated GBE1 stable-overexpression or knockdown PC cells. Cell Counting Kit (CCK-8), colony formation assay, sphere formation assay and flow cytometry assay were conducted to analyze cell proliferation and stemness ability in vitro. Subcutaneous and orthotopic mouse models were used to verify the function of GBE1 in vivo. RNA immunoprecipitation (RIP) assay, RNA stability experiment and western blots were conducted to explore the molecular regulation of GBE1 in PC.
RESULTS: GBE1 was significantly upregulated in PC and associated with poor prognosis of PC patients. Functionally, GBE1 overexpression facilitated PC cell proliferation and stemness-like properties, while knockdown of GBE1 attenuated the malignancy of PC cells. Importantly, we found the m6A modification of GBE1 RNA, and WTAP and IGF2BP3 was revealed as the m6A regulators to increase GBE1 mRNA stability and expression. Furthermore, c-Myc was discovered as a downstream gene of GBE1 and functional rescue experiments showed that overexpression of c-Myc could rescue GBE1 knockdown-induced PC cell growth inhibition.
CONCLUSIONS: Our study uncovered the oncogenic role of GBE1/c-Myc axis in PC progression and revealed WTAP/IGF2BP3-mediated m6A modification of GBE1, which highlight the potential application of GBE1 in the targeted therapy of PC.

Renal aging and the subsequent rise in kidney-related diseases are attributed to senescence in renal tubular epithelial cells (RTECs). Our study revealed that the abnormal expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), a reader of RNA N6-methyladenosine, is critically involved in cisplatin-induced renal tubular senescence. In cisplatin-induced senescence of RTECs, the promoter activity and transcription of IGF2BP3 is markedly suppressed. It was due to the down regulation of MYC proto-oncogene (MYC), which regulates IGF2BP3 transcription by binding to the putative site at 1852-1863 of the IGF2BP3 promoter. Overexpression of IGF2BP3 ameliorated cisplatin-induced renal tubular senescence in vitro. Mechanistic studies revealed that IGF2BP3 inhibits cellular senescence in RTECs by enhancing cyclin-dependent kinase 6 (CDK6) mRNA stability and increasing its expression. The inhibition effect of IGF2BP3 on tubular senescence is partially reversed by the knockdown of CDK6. Further, IGF2BP3 recruits nuclear cap binding protein subunit 1 (NCBP1) and inhibits CDK6 mRNA decay, by recognizing m6A modification. Specifically, IGF2BP3 recognizes m6A motif \"GGACU\" at nucleotides 110-114 in the 5\' untranslated region (UTR) field of CDK6 mRNA. The involvement of IGF2BP3/CDK6 in alleviating tubular senescence was confirmed in a cisplatin-induced acute kidney injury (AKI)-to-chronic kidney disease (CKD) model. Clinical data also suggests an age-related decrease in IGF2BP3 and CDK6 levels in renal tissue or serum samples from patients. These findings suggest that IGF2BP3/CDK6 may be a promising target in cisplatin-induced tubular senescence and renal failure.

N6-methyladenosine (m6A) methylation plays a crucial role in various biological processes and the pathogenesis of human diseases. However, its role and mechanism in kidney fibrosis remain elusive. In this study, we show that the overall level of m6A methylated RNA was upregulated and the m6A methyltransferase METTL3 was induced in kidney tubular epithelial cells in mouse models and human kidney biopsies of chronic kidney disease (CKD). Proximal tubule-specific knockout of METTL3 in mice protected kidneys against developing fibrotic lesions after injury. Conversely, overexpression of METTL3 aggravated kidney fibrosis in vivo. Through bioinformatics analysis and experimental validation, we identified β-catenin mRNA as a major target of METTL3-mediated m6A modification, which could be recognized by a specific m6A reader, the insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3). METTL3 stabilized β-catenin mRNA, increased β-catenin protein and induced its downstream profibrotic genes, whereas either knockdown of IGF2BP3 or inhibiting β-catenin signaling abolished its effects. Collectively, these results indicate that METTL3 promotes kidney fibrosis by stimulating the m6A modification of β-catenin mRNA, leading to its stabilization and its downstream profibrotic genes expression. Our findings suggest that targeting METTL3/IGF2BP3/β-catenin pathway may be a novel strategy for the treatment of fibrotic CKD.

Understanding the molecular characteristics of triple-negative breast cancer (TNBC) and developing more tailored treatment approaches is crucial. Circular RNAs (circRNAs), as potential therapeutic targets, remain largely unexplored in TNBC. This study utilized circRNA microarray analysis to determine the expression of circRNAs in TNBC, analyzing nine patient specimens. The characteristics of circBRAF were examined using divergent PCR primers, Sanger sequencing, fluorescence in situ hybridization (FISH) analysis, and the application of RNase and actinomycin D. The biological function of circBRAF in TNBC was further investigated through colony formation, tube formation, and transwell assays. Crucially, the mechanisms underlying the effects of circBRAF on TNBC progression were explored via RNA immunoprecipitation sequencing (RIP-seq) data, MS2 pulldown, RNA sequencing (RNA-seq) analysis, circBRAF knockdown, histone H3K9me3 modification, and Chromatin Isolation by RNA Purification (ChIRP) tests followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We focused particularly on hsa_circ_0007178, produced from exons 4-13 of the oncogene BRAF. Functional experiments revealed that circBRAF is crucial for the development of TNBC, with its knockdown preventing angiogenesis, metastasis, and cell division in vitro. Mechanistically, circBRAF interacts with KDM4B and IGF2BP3, promoting TNBC growth. Interaction of circBRAF with IGF2BP3 increased the expression of VCAN, FN1, CDCA3, or B4GALT3 by controlling mRNA stability through RNA N6-methyladenosine (m6A) modification. Furthermore, circBRAF upregulated the expression of ADAMTS14 and MMP9 through recruitment of KDM4B to enhance respective H3K9me3 modification. Furthermore, overexpression of circBRAF was able to overcome the inhibitory effects of siKDM4B and siIGF2BP3 on cell migration and invasion. Our findings suggest that circBRAF may act as an oncogene in TNBC through its specific interactions with KDM4B and IGF2BP3, implying that circBRAF could serve as a potentially effective novel therapeutic target for TNBC.

BACKGROUND: Breast cancer is the most common malignant tumor, and metastasis remains the major cause of poor prognosis. Glucose metabolic reprogramming is one of the prominent hallmarks in cancer, providing nutrients and energy to support dramatically elevated tumor growth and metastasis. Nevertheless, the potential mechanistic links between glycolysis and breast cancer progression have not been thoroughly elucidated.
METHODS: RNA-seq analysis was used to identify glucose metabolism-related circRNAs. The expression of circSIPA1L3 in breast cancer tissues and serum was examined by qRT-PCR, and further assessed its diagnostic value. We also evaluated the prognostic potential of circSIPA1L3 by analyzing a cohort of 238 breast cancer patients. Gain- and loss-of-function experiments, transcriptomic analysis, and molecular biology experiments were conducted to explore the biological function and regulatory mechanism of circSIPA1L3.
RESULTS: Using RNA-seq analysis, circSIPA1L3 was identified as the critical mediator responsible for metabolic adaption upon energy stress. Gain- and loss-of-function experiments revealed that circSIPA1L3 exerted a stimulative effect on breast cancer progression and glycolysis, which could also be transported by exosomes and facilitated malignant behaviors among breast cancer cells. Significantly, the elevated lactate secretion caused by circSIPA1L3-mediated glycolysis enhancement promoted the recruitment of tumor associated macrophage and their tumor-promoting roles. Mechanistically, EIF4A3 induced the cyclization and cytoplasmic export of circSIPA1L3, which inhibited ubiquitin-mediated IGF2BP3 degradation through enhancing the UPS7-IGF2BP3 interaction. Furthermore, circSIPA1L3 increased mRNA stability of the lactate export carrier SLC16A1 and the glucose intake enhancer RAB11A through either strengthening their interaction with IGF2BP3 or sponging miR-665, leading to enhanced glycolytic metabolism. Clinically, elevated circSIPA1L3 expression indicated unfavorable prognosis base on the cohort of 238 breast cancer patients. Moreover, circSIPA1L3 was highly expressed in the serum of breast cancer patients and exhibited high diagnostic value for breast cancer patients.
CONCLUSIONS: Our study highlights the oncogenic role of circSIPA1L3 through mediating glucose metabolism, which might serve as a promising diagnostic and prognostic biomarker and potential therapeutic target for breast cancer.

BACKGROUND: Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) has been implicated in numerous inflammatory and cancerous conditions. However, its precise molecular mechanisms in endometriosis (EMs) remains unclear. The aim of this study is to examine the influence of IGF2BP3 on the occurrence and progression of EMs and to elucidate its underlying molecular mechanism.
METHODS: Efects of IGF2BP3 on endometriosis were confrmed in vitro and in vivo. Based on bioinformatics analysis, RNA immunoprecipitation (RIP), RNA pull-down assays and Fluorescent in situ hybridization (FISH) were used to show the association between IGF2BP3 and UCA1. Single-cell spatial transcriptomics analysis shows the expression distribution of glutaminase 1 (GLS1) mRNA in EMs. Study the effect on glutamine metabolism after ectopic endometriotic stromal cells (eESCs) were transfected with Sh-IGF2BP3 and Sh-UCA1 lentivirus.
RESULTS: Immunohistochemical staining have revealed that IGF2BP3 was upregulated in ectopic endometriotic lesions (EC) compared to normal endometrial tissues (EN). The proliferation and migration ability of eESCs were greatly reduced by downregulating IGF2BP3. Additionally, IGF2BP3 has been observed to interact with urothelial carcinoma associated 1 (UCA1), leading to increased stability of GLS1 mRNA and subsequently enhancing glutamine metabolism. Results also demonstrated that IGF2BP3 directly interacts with the 3\' UTR region of GLS1 mRNA, influencing its expression and stability. Furthermore, UCA1 was able to bind with c-MYC protein, stabilizing c-MYC mRNA and consequently enhancing GLS1 expression through transcriptional promotion.
CONCLUSIONS: These discoveries underscored the critical involvement of IGF2BP3 in the elevation and stability of GLS1 mRNA in the context of glutamine metabolism by interacting with UCA1 in EMs. The implications of our study extended to the identification of possible therapeutic targets for individuals with EMs.

BACKGROUND: Berberine (BBR), an isoquinoline alkaloid from Coptidis rhizoma, has been found to have powerful activities against various human malignancies, including breast cancer. However, the underlying antitumor mechanisms of BBR in breast cancer remain poorly understood.
METHODS: Breast cancer cells were cultured and treated with different doses (0, 20, 40, and 60 μM) of BBR for 48 h. Cell viability, proliferation, apoptosis, invasion, and migration were assessed using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), 5-ethynyl-2\'-deoxyuridine (EdU), flow cytometry, transwell, and wound healing assays. Fibroblast growth factor 7 (FGF7), methyltransferase-like 3 (METTL3), and insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) mRNA levels and protein levels were measured using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Interaction between METTL3 and FGF7 m6A was assessed using methylated RNA immunoprecipitation (MeRIP)-qPCR and RNA immunoprecipitation (RIP) assay. Binding ability between IGF2BP3 and FGF7 mRNA was analyzed using RIP assay.
RESULTS: BBR treatment hindered breast cancer cell proliferation, invasion, migration, and induced apoptosis. FGF7 expression was upregulated in breast cancer tissues, while its level was reduced in BBR-treated tumor cells. FGF7 upregulation relieved the repression of BBR on breast cancer cell malignant behaviors. In mechanism, METTL3 stabilized FGF7 mRNA through the m6A-IGF2BP3-dependent mechanism and naturally improved FGF7 expression. BBR treatment inhibited breast cancer growth in vivo.
CONCLUSIONS: BBR treatment blocked breast cancer cell growth and metastasis partly by regulating METTL3-mediated m6A modification of FGF7 mRNA, providing a promising therapeutic target for breast cancer treatment.

The objective of this study was to better characterize the role of the glutamine transporter SLC38A1 in cervical cancer and explore the underlying mechanisms. Data from public databases and clinical cervical cancer tissue samples were used to assess the expression of SLC38A1 and its prognostic significance. Immunohistochemical staining, qRT-PCR, and Western blotting were used to evaluate the expression of relevant genes and proteins. Cell viability, cell cycle, apoptosis, and intracellular glutamine content were measured using CCK-8, flow cytometry, and biochemical assays. Additionally, the RNA immunoprecipitation (RIP) assay was used to examine the impact of METTL3/IGF2BP3 on the m6A modification of the SLC38A1 3\'UTR. Both cervical cancer specimens and cells showed significantly increased expression of SLC38A1 and its expression correlated with an unfavorable prognosis. Knockdown of SLC38A1 inhibited cell viability and cell cycle progression, induced apoptosis, and suppressed tumor growth in vivo. Glutaminase-1 inhibitor CB-839 reversed the effects of SLC38A1 overexpression. METTL3 promoted m6A modification of SLC38A1 and enhanced its mRNA stability through IGF2BP3 recruitment. Moreover, METTL3 silencing inhibited cell viability, cell cycle progression, intracellular glutamine content, and induced apoptosis, but these effects were reversed by SLC38A1 overexpression. In conclusion, METTL3-mediated m6A methylation of SLC38A1 stimulates cervical cancer progression. SLC38A1 inhibition is a potential therapeutic strategy for cervical cancer.

Numerous studies have characterized the critical role of circular RNAs (circRNAs) as regulatory factors in the progression of multiple cancers. However, the biological functions of circRNAs and their underlying molecular mechanisms in the progression of uveal melanoma (UM) remain enigmatic. In this study, we identified a novel circRNA, circ_0053943, through re-analysis of UM microarray data and quantitative RT-PCR. Circ_0053943 was found to be upregulated in UM and to promote the proliferation and metastatic ability of UM cells in both in vitro and in vivo settings. Mechanistically, circ_0053943 was observed to bind to the KH1 and KH2 domains of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), thereby enhancing the function of IGF2BP3 by stabilizing its target mRNA. RNA sequencing assays identified epidermal growth factor receptor (EGFR) as a target gene of circ_0053943 and IGF2BP3 at the transcriptional level. Rescue assays demonstrated that circ_0053943 exerts its biological function by stabilizing EGFR mRNA and regulating the downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway. Collectively, circ_0053943 may promote UM progression by stabilizing EGFR mRNA and activating the MAPK/ERK signaling pathway through the formation of a circ_0053943/IGF2BP3/EGFR RNA-protein ternary complex, thus providing a potential biomarker and therapeutic target for UM.

Human papillomavirus (HPV) infection has been reported to be associated with N6-methyladenosine (m6A) modification in cancers. However, the underlying mechanism by which m6A methylation participates in HPV-related cervical squamous cell carcinoma (CSCC) remains largely unclear. In this study, we observed that m6A regulators methyltransferase like protein (METTL14) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) were upregulated in HPV-positive CSCC tissues and cell lines, and their high expression predicted poor prognosis for HPV-infected CSCC patients. Cellular functional experiments verified that HPV16 oncogenes E6/E7 upregulated the expression of METTL14 and IGF2BP3 to promote cell proliferation and epithelial mesenchymal transition of CSCC cells. Next, we found that E6/E7 stabilized fascin actin-bundling protein 1 (FSCN1) mRNA and elevated FSCN1 expression in CSCC cells through upregulating METTL14/IGF2BP3-mediated m6A modification, and FSCN1 expression was also validated to be positively associated with worse outcomes of HPV-positive CSCC patients. Finally, HPV16-positive CSCC cell lines SiHa and CaSki were transfected with knockdown vector for E6/E7 or METTL14/IGF2BP3 and overexpressing vector for FSCN1, and functional verification experiments were performed through using MTT assay, flow cytometry, wound healing assay and tumour formation assay. Results indicated that knockdown of E6/E7 or METTL14/IGF2BP3 suppressed cell proliferation, migration and tumorigenesis, and accelerated cell apoptosis of HPV-positive CSCC cells. Their tumour-suppressive effects were abolished through overexpressing FSCN1. Overall, HPV E6/E7 advanced CSCC development through upregulating METTL14/IGF2BP3-mediated FSCN1 m6A modification.