Perineural invasion (PNI) is a biological characteristic commonly observed in pancreatic cancer. Although PNI plays a key role in pancreatic cancer metastasis, recurrence, and poor postoperative survival, its mechanism is largely unclarified. Clinical sample analysis and endoscopic ultrasonographic elasticity scoring indicated that cancer-associated fibroblasts (CAFs) were closely related to the occurrence of PNI. Furthermore, CAF-derived extracellular vesicles (EVs) were involved in PNI in dorsal root ganglion coculture and mouse sciatic nerve models. Next, we demonstrated that CAFs promoted PNI through extracellular vesicle transmission of PNI-associated transcript (PIAT). Mechanistically, PIAT specifically bound to YBX1 and blocked the YBX1-Nedd4l interaction to inhibit YBX1 ubiquitination and degradation. Furthermore, PIAT enhanced the binding of YBX1 and PNI-associated mRNAs in a 5-methylcytosine (m5C)-dependent manner. Mutation of m5C recognition motifs in YBX1 or m5C sites in downstream target genes reversed PIAT-mediated PNI. Consistent with these findings, analyses using a KPC mouse model demonstrated that the PIAT/YBX1 axis enhanced PNI through m5C modification. Clinical data suggested that the PIAT expression in the serum EVs of patients with pancreatic cancer was associated with the degree of neural invasion and prognosis. Our study revealed the important role of the PIAT/YBX1 signaling axis in the tumor microenvironment (TME) in promoting tumor cell PNI and provided a new target for precise interference with CAFs and RNA methylation in the TME to suppress PNI in pancreatic cancer.

Ewing\'s sarcoma (ES) represents a rare yet exceedingly aggressive neoplasm that poses a significant health risk to the pediatric and adolescent population. The clinical outcomes for individuals with relapsed or refractory ES are notably adverse, primarily attributed to the constrained therapeutic alternatives available. Despite significant advancements in the field, molecular pathology-driven therapeutic strategies have yet to achieve a definitive reduction in the mortality rates associated with ES. Consequently, there exists an imperative need to discover innovative therapeutic targets to effectively combat ES. To reveal the mechanism of the SETD8 (also known as lysine methyltransferase 5A) inhibitor UNC0379, cell death manners were analyzed with different inhibitors. The contributions of SETD8 to the processes of apoptosis and ferroptosis in ES cells were evaluated employing the histone methyltransferase inhibitor UNC0379 in conjunction with RNA interference techniques. The molecular regulatory mechanisms of SETD8 in ES were examined through the application of RNA sequencing (RNA-seq) and mass spectrometry-based proteomic analysis. Moreover, nude mouse xenograft models were established to explore the role of SETD8 in ES in vivo. SETD8, a sole nucleosome-specific methyltransferase that catalyzes mono-methylation of histone H4 at lysine 20 (H4K20me1), was found to be upregulated in ES, and its overexpression was associated with dismal outcomes of patients. SETD8 knockdown dramatically induced the apoptosis and ferroptosis of ES cells in vitro and suppressed tumorigenesis in vivo. Mechanistic investigations revealed that SETD8 facilitated the nuclear translocation of YBX1 through post-transcriptional regulatory mechanisms, which subsequently culminated in the transcriptional upregulation of RAC3. In summary, SETD8 inhibits the apoptosis and ferroptosis of ES cells through the YBX1/RAC3 axis, which provides new insights into the mechanism of tumorigenesis of ES. SETD8 may be a potential target for clinical intervention in ES patients.

BACKGROUND: Lysine methyltransferase 2D (KMT2D) mediates mono-methylation of histone H3 lysine 4 (H3K4me1) in mammals. H3K4me1 mark is involved in establishing an active chromatin structure to promote gene transcription. However, the precise molecular mechanism underlying the KMT2D-mediated H3K4me1 mark modulates gene expression in triple-negative breast cancer (TNBC) progression is unresolved.
RESULTS: We recognized Y-box-binding protein 1 (YBX1) as a \"reader\" of the H3K4me1 mark, and a point mutation of YBX1 (E121A) disrupted this interaction. We found that KMT2D and YBX1 cooperatively promoted cell growth and metastasis of TNBC cells in vitro and in vivo. The expression levels of KMT2D and YBX1 were both upregulated in tumour tissues and correlated with poor prognosis for breast cancer patients. Combined analyses of ChIP-seq and RNA-seq data indicated that YBX1 was co-localized with KMT2D-mediated H3K4me1 in the promoter regions of c-Myc and SENP1, thereby activating their expressions in TNBC cells. Moreover, we demonstrated that YBX1 activated the expressions of c-Myc and SENP1 in a KMT2D-dependent manner.
CONCLUSIONS: Our results suggest that KMT2D-mediated H3K4me1 recruits YBX1 to facilitate TNBC progression through epigenetic activation of c-Myc and SENP1. These results together unveil a crucial interplay between histone mark and gene regulation in TNBC progression, thus providing novel insights into targeting the KMT2D-H3K4me1-YBX1 axis for TNBC treatment.
CONCLUSIONS: YBX1 is a KMT2D-mediated H3K4me1-binding effector protein and mutation of YBX1 (E121A) disrupts its binding to H3K4me1. KMT2D and YBX1 cooperatively promote TNBC proliferation and metastasis by activating c-Myc and SENP1 expression in vitro and in vivo. YBX1 is colocalized with H3K4me1 in the c-Myc and SENP1 promoter regions in TNBC cells and increased YBX1 expression predicts a poor prognosis in breast cancer patients.

BACKGROUND: The vast majority of lncRNAs have low expression abundance, which greatly limits their functional range and impact. As a high expression abundance lncRNA, FGD5-AS1\'s non-ceRNA biological function in cancer is unclear.
METHODS: RNA-seq studies and chromatin immunoprecipitation (Chip) assays were performed to identify ZEB1-regulated lncRNAs. RNA sequencing, RNA pulldown, RNA Immunoprecipitation assays, and rescue assays were conducted to explore the molecular mechanisms of FGD5-AS1 in GC.
RESULTS: As one of the most abundant lncRNAs in cells, FGD5-AS1 has been shown to be transcriptionally activated by ZEB1, thus closely related to epithelial-mesenchymal transition (EMT) signaling. Clinical analysis showed that FGD5-AS1 overexpression was clinically associated with lymph node metastasis, and predicted poor survival in GC. Loss-of-function studies confirmed that FGD5-AS1 knockdown inhibited GC proliferation and induced cisplatin chemosensibility, cell senescence, and DNA damage in GC cells. Mechanismically, FGD5-AS1 is a YBX1-binding lncRNA due to its mRNA contains three adjacent structural motifs (UAAUCCCA, ACCAGCCU, and CAGUGAGC) that can be recognized and bound by YBX1. And this RNA-protein interaction prolonged the half-life of the YBX1 protein in GC. Additionally, a rescue assay showed that FGD5-AS1 promotes GC by repressing cell senescence and ROS production via YBX1.
CONCLUSIONS: FGD5-AS1 is a cellular high-abundant lncRNA that is transcriptionally regulated by ZEB1. FGD5-AS1 overexpression promoted GC progression by inhibiting cell senescence and ROS production through binding and stabilizing the YBX1 protein.

Hepatitis C virus (HCV) is a positive-stranded RNA virus that mainly causes chronic hepatitis, cirrhosis and hepatocellular carcinoma. Recently we confirmed m5C modifications within NS5A gene of HCV RNA genome. However, the roles of the m5C modification and its interaction with host proteins in regulating HCV\'s life cycle, remain unexplored. Here, we demonstrate that HCV infection enhances the expression of the host m5C reader YBX1 through the transcription factor MAX. YBX1 acts as an m5C reader, recognizing the m5C-modified NS5A C7525 site in the HCV RNA genome and significantly enhancing HCV RNA stability. This m5C-modification is also required for YBX1 colocalization with lipid droplets and HCV Core protein. Moreover, YBX1 facilitates HCV RNA replication, as well as viral assembly/budding. The tryptophan residue at position 65 (W65) of YBX1 is critical for these functions. Knockout of YBX1 or the application of YBX1 inhibitor SU056 suppresses HCV RNA replication and viral protein translation. To our knowledge, this is the first report demonstrating that the interaction between host m5C reader YBX1 and HCV RNA m5C methylation facilitates viral replication. Therefore, hepatic-YBX1 knockdown holds promise as a potential host-directed strategy for HCV therapy.

Rationale: Current treatments for ocular angiogenesis primarily focus on blocking the activity of vascular endothelial growth factor (VEGF), but unfavorable side effects and unsatisfactory efficacy remain issues. The identification of novel targets for anti-angiogenic treatment is still needed. Methods: We investigated the role of tsRNA-1599 in ocular angiogenesis using endothelial cells, a streptozotocin (STZ)-induced diabetic model, a laser-induced choroidal neovascularization model, and an oxygen-induced retinopathy model. CCK-8 assays, EdU assays, transwell assays, and matrigel assays were performed to assess the role of tsRNA-1599 in endothelial cells. Retinal digestion assays, Isolectin B4 (IB4) staining, and choroidal sprouting assays were conducted to evaluate the role of tsRNA-1599 in ocular angiogenesis. Transcriptomic analysis, metabolic analysis, RNA pull-down assays, and mass spectrometry were utilized to elucidate the mechanism underlying angiogenic effects mediated by tsRNA-1599. Results: tsRNA-1599 expression was up-regulated in experimental ocular angiogenesis models and endothelial cells in response to angiogenic stress. Silencing of tsRNA-1599 suppressed angiogenic effects in endothelial cells in vitro and inhibited pathological ocular angiogenesis in vivo. Mechanistically, tsRNA-1599 exhibited little effect on VEGF signaling but could cause reduced glycolysis and NAD+/NADH production in endothelial cells by regulating the expression of HK2 gene through interacting with YBX1, thus affecting endothelial effects. Conclusions: Targeting glycolytic reprogramming of endothelial cells by a tRNA-derived small RNA represents an exploitable therapeutic approach for ocular neovascular diseases.

Platinum-based chemotherapy causes genetic damage and induces apoptosis in ovarian cancer cells. Enhancing the ability to resist platinum drug-induced DNA damage and apoptotic stress is critical for tumor cells to acquire drug resistance. Here, we found that Y-box binding protein 1 (YBX1) was highly expressed in cisplatin-resistant patient-derived organoids (PDOs) and was a crucial gene for alleviating platinum-induced stress and maintaining drug resistance characteristics in ovarian cancer cells. Mechanistically, YBX1 recognized m5C modifications in CHD3 mRNA and maintained mRNA stability by recruiting PABPC1 protein. This regulatory process enhanced chromatin accessibility and improved the efficiency of homologous recombination (HR) repair, facilitating tumor cells to withstand platinum-induced apoptotic stress. In addition, SU056, an inhibitor of YBX1, exhibited the potential to reverse platinum resistance in subcutaneous and PDO orthotopic xenograft models. In conclusion, YBX1 is critical for ovarian cancer cells to alleviate the platinum-induced stress and may be a potential target for reversing drug-resistant therapies.

BACKGROUND: RNA m5C methylation has been extensively implicated in the occurrence and development of tumors. As the main methyltransferase, NSUN2 plays a crucial regulatory role across diverse tumor types. However, the precise impact of NSUN2-mediated m5C modification on breast cancer (BC) remains unclear. Our study aims to elucidate the molecular mechanism underlying how NSUN2 regulates the target gene HGH1 (also known as FAM203) through m5C modification, thereby promoting BC progression. Additionally, this study targets at preliminarily clarifying the biological roles of NSUN2 and HGH1 in BC.
METHODS: Tumor and adjacent tissues from 5 BC patients were collected, and the m5C modification target HGH1 in BC was screened through RNA sequencing (RNA-seq) and single-base resolution m5C methylation sequencing (RNA-BisSeq). Methylation RNA immunoprecipitation-qPCR (MeRIP-qPCR) and RNA-binding protein immunoprecipitation-qPCR (RIP-qPCR) confirmed that the methylation molecules NSUN2 and YBX1 specifically recognized and bound to HGH1 through m5C modification. In addition, proteomics, co-immunoprecipitation (co-IP), and Ribosome sequencing (Ribo-Seq) were used to explore the biological role of HGH1 in BC.
RESULTS: As the main m5C methylation molecule, NSUN2 is abnormally overexpressed in BC and increases the overall level of RNA m5C. Knocking down NSUN2 can inhibit BC progression in vitro or in vivo. Combined RNA-seq and RNA-BisSeq analysis identified HGH1 as a potential target of abnormal m5C modifications. We clarified the mechanism by which NSUN2 regulates HGH1 expression through m5C modification, a process that involves interactions with the YBX1 protein, which collectively impacts mRNA stability and protein synthesis. Furthermore, this study is the first to reveal the binding interaction between HGH1 and the translation elongation factor EEF2, providing a comprehensive understanding of its ability to regulate transcript translation efficiency and protein synthesis in BC cells.
CONCLUSIONS: This study preliminarily clarifies the regulatory role of the NSUN2-YBX1-m5C-HGH1 axis from post-transcriptional modification to protein translation, revealing the key role of abnormal RNA m5C modification in BC and suggesting that HGH1 may be a new epigenetic biomarker and potential therapeutic target for BC.

BACKGROUND: Neuroinflammation is a characteristic pathological change of Alzheimer\'s Diseases (AD). Microglia have been reported to participate in inflammatory responses within the central nervous system. However, the mechanism of microglia released exosome (EXO) contribute to communication within AD microenvironment remains obscure.
METHODS: The interaction between microglia and AD was investigated in vitro and in vivo. RNA-binding protein immunoprecipitation (RIP) was used to investigate the mechanisms of miR-223 and YB-1. The association between microglia derived exosomal YB-1/miR-223 axis and nerve cell damage were assessed using Western blot, immunofluorescence, RT-PCR, ELISA and wound healing assay.
RESULTS: Here, we reported AD model was responsible for the M1-like (pro-inflammatory) polarization of microglia which in turn induced nerve cell damage. While M2-like (anti-inflammatory) microglia could release miR-223-enriched EXO which reduced neuroinflammation and ameliorated nerve damage in AD model in vivo and in vitro. Moreover, YB-1 directly interacted with miR-223 both in cell and EXO, and participated in microglia exosomal miR-223 loading.
CONCLUSIONS: These results indicate that anti-inflammatory microglia-mediated neuroprotection form inflammatory damage involves exporting miR-223 via EXO sorted by YB-1. Consequently, YB-1-mediated microglia exosomal sorting of miR-223 improved the nerve cell damage repair, representing a promising therapeutic target for AD.

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. Mesenchymal stem cells-derived small extracellular vesicles (MSCs-sEVs) are attractive candidates for ovarian function restoration and folliculogenesis for POF due to their safety and efficacy, however, the key mediator in MSCs-sEVs that modulates this response and underlying mechanisms remains elusive. Herein, we reported that YB-1 protein was markedly downregulated in vitro and in vivo models of POF induced with H2O2 and CTX respectively, accompanied by granulosa cells (GCs) senescence phenotype. Notably, BMSCs-sEVs transplantation upregulated YB-1, attenuated oxidative damage-induced cellular senescence in GCs, and significantly improved the ovarian function of POF rats, but that was reversed by YB-1 depletion. Moreover, YB-1 showed an obvious decline in serum and GCs in POF patients. Mechanistically, YB-1 as an RNA-binding protein (RBP) physically interacted with a long non-coding RNA, MALAT1, and increased its stability, further, MALAT1 acted as a competing endogenous RNA (ceRNA) to elevate FOXO3 levels by sequestering miR-211-5p to prevent its degradation, leading to repair of ovarian function. In summary, we demonstrated that BMSCs-sEVs improve ovarian function by releasing YB-1, which mediates MALAT1/miR-211-5p/FOXO3 axis regulation, providing a possible therapeutic target for patients with POF.