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N6-methyladenosine (m6A) modification is, a more common epigenetic modification, mainly found in mRNA. More and more researches have shown the important functions of m6A on human cancers. This study seeks to explore the role of hnRNPA2B1 and m6A-dependent mechanism in cervical cancer. Elevated hnRNPA2B1 indicated the poor prognosis of cervical cancer patients. Enforced hnRNPA2B1 reduced the apoptosis, and accelerated the proliferation and migration of cervical cancer cells in vitro. Besides, hnRNPA2B1 promoted the aerobic glycolysis of cervical cancer cells, including the lactate secretion, glucose uptake, ATP production, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). LDHA was found as the downstream target of hnRNPA2B1 by m6A site. Moreover, hnRNPA2B1 enhanced the mRNA stability of LDHA through m6A-dependent manner. LDHA inhibitor (FX-11) could reverse the effect of hnRNPA2B1. Taken together, the data revealed that hnRNPA2B1 promoted the proliferation, migration and aerobic glycolysis of cervical cancer cells by m6A/LDHA-dependent manner. These findings might bring a new idea for cervical cancer treatment.
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BACKGROUND: Multiple myeloma (MM), characterized with bone marrow microenvironment disorder, accounts for about 20% of hematological cancer deaths globally. Tissue extracellular communication, especially extracellular vesicles, has been defined as important mediator among cell-to-cell cross-talk. Our previous study revealed an elevated level of H19 in MM, whereas, its role in MM exosomes in the development of osteolysis remains largely unknown. METHOD: MM exosomes referring to 5TGM1 cells were isolated and characterized using transmission electron microscopy (TEM), nanoparticle tracking and western blot analysis. The biological effects of blocking H19 were examined on osteolysis in vivo of C57Bl6/KalwRij mice, as well as on the osteoclast differentiation in vitro of RAW264.7 cells, by the application of TRAP, either with osteogenic differentiation in vitro of bone marrow mesenchymal stem cells (BMSCs), by the detection of alkaline phosphatase (ALP), alizarin red dye staining (ARS). The targeted relationships among H19/hnRNPA2B1/BET proteins were validated through RNA immunoprecipitation (RIP) and RNA pull-down assays. RESULTS: 5TGM1 cells derived-exosomes lacking H19 dramatically blocked osteolysis and boosted osteogeneis in C57Bl6/KalwRij mice, either with osteoclastic differentiation of RAW264.7 cells and osteogenic differentiation of BMSCs, thereby enhancing their resorptive activity. Physically, H19 interacted with hnRNPA2B1 by preferentially adhering to it and enhancing its nuclear-cytoplasmic translocation. Further mechanistic research validated that H19 promoted the stabilization of BET proteins through hnRNA2B1 to be involved in osteoclast differentiation for contributing to MM progression. CONCLUSION: Altogether, our findings suggest that H19, serving as an essential role for exosomes in the bone marrow environment, might be a viable diagnostic and therapeutic target for MM therapy.
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Exosomas , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Ratones Endogámicos C57BL , Mieloma Múltiple , Osteoblastos , Osteoclastos , ARN Largo no Codificante , Animales , Mieloma Múltiple/patología , Mieloma Múltiple/metabolismo , Osteoclastos/metabolismo , Ratones , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/genética , Células RAW 264.7 , Exosomas/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Osteoblastos/metabolismo , Humanos , Diferenciación Celular , Línea Celular Tumoral , Osteólisis/metabolismo , Osteogénesis , Células Madre Mesenquimatosas/metabolismoRESUMEN
Recent studies have confirmed that melatonin and N6-methyladenosine (m6A) modification can influence bone cell differentiation and bone formation. Melatonin can also regulate a variety of biological processes through m6A modification. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) serves as a reader of m6A modification. In this study, we used the hindlimb unloading model as an animal model of bone loss induced by simulated microgravity and used 2D clinorotation to simulate a microgravity environment for cells on the ground. We found that hnRNPA2B1 was downregulated both in vitro and in vivo during simulated microgravity. Further investigations showed that hnRNPA2B1 could promote osteoblast differentiation and that overexpression of hnRNPA2B1 attenuated the suppression of osteoblast differentiation induced by simulated microgravity. We also discovered that melatonin could promote the expression of hnRNPA2B1 under simulated microgravity. Moreover, we found that promotion of osteoblast differentiation by melatonin was partially dependent on hnRNPA2B1. Therefore, this research revealed, for the first time, the role of the melatonin/hnRNPA2B1 axis in osteoblast differentiation under simulated microgravity. Targeting this axis may be a potential protective strategy against microgravity-induced bone loss and osteoporosis.
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BACKGROUND: Numerous studies have shown that m6A plays an important regulatory role in the development of tumors. HNRNPA2B1, one of the m6A RNA methylation reading proteins, has been proven to be elevated in human cancers. OBJECTIVE: In this study, we aimed to identify the role of HNRNPA2B1 in breast cancer. METHODS: HNRNPA2B1 expression was investigated via RT-qPCR and TCGA database in breast cancer. Then, the function of HNRNPA2B1 on cancer cell was measured by CCK8 assays, colony formation and scratch assays. In addition, HNRNPA2B1 expression in BRCA was explored via the Wilcoxon signed-rank test, KruskalWallis test and logistic regression. The association with HNRNPA2B1 expression and survival were considered by KaplanMeier and Cox regression analyses. The biological function of HNRNPA2B1 was analyzed via gene set enrichment analysis (GSEA) and the cluster Profiler R software package. RESULTS: We found that HNRNPA2B1 was highly expressed and induced cell proliferation and migration in breast cancer. Moreover, we observed HNRNPA2B1 induced tumor growth in vivo. In addition, we also found HNRNPA2B1 expression was associated with characteristics and prognosis in breast cancer patients. CONCLUSION: Our findings suggested that HNRNPA2B1 promoted tumor growth and could function as a new potential molecular marker in breast cancer.
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Biomarcadores de Tumor , Neoplasias de la Mama , Proliferación Celular , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Animales , Femenino , Humanos , Ratones , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , PronósticoRESUMEN
Viruses deploy sophisticated strategies to hijack the host's translation machinery to favor viral protein synthesis and counteract innate cellular defenses. However, little is known about the mechanisms by which Senecavirus A (SVA) controls the host's translation. Using a series of sophisticated molecular cell manipulation techniques, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as an essential host factor involved in translation control in SVA-infected cells. It was also determined that the SVA structural protein, VP3, binds to and relocalizes hnRNPA2B1, which interferes with the host's protein synthesis machinery to establish a cellular environment that facilitates viral propagation via a two-pronged strategy: first, hnRNPA2B1 serves as a potent internal ribosome entry site (IRES) trans-acting factor, which is selectively co-opted to promote viral IRES-driven translation by supporting the assembly of translation initiation complexes. Second, a strong repression of host cell translation occurs in the context of the VP3-hnRNPA2B1 interaction, resulting in attenuation of the interferons response. This is the first study to demonstrate the interaction between SVA VP3 and hnRNPA2B1, and to characterize their key roles in manipulating translation. This novel dual mechanism, which regulates selective mRNA translation and immune evasion of virus-infected cells, highlights the VP3-hnRNPA2B1 complex as a potential target for the development of modified antiviral or oncolytic reagents. IMPORTANCE: Viral reproduction is contingent on viral protein synthesis, which relies entirely on the host's translation machinery. As such, viruses often need to control the cellular translational apparatus to favor viral protein production and avoid host innate defenses. Senecavirus A (SVA) is an important virus, both as an emerging pathogen in the pork industry and as a potential oncolytic virus for neuroendocrine cancers. Here, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as a critical regulator of the translational landscape during SVA infection. This study supports a model whereby the VP3 protein of SVA efficiently subverts the host's protein synthesis machinery through its ability to bind to and relocalize hnRNPA2B1, not only selectively promoting viral internal ribosome entry site-driven translation but also resulting in global translation shutdown and immune evasion. Together, these data provide new insights into how the complex interactions between translation machinery, SVA, and innate immunity contribute to the pathogenicity of the SVA.
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Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Inmunidad Innata , Sitios Internos de Entrada al Ribosoma , Picornaviridae , Biosíntesis de Proteínas , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Humanos , Picornaviridae/inmunología , Interacciones Huésped-Patógeno/inmunología , Células HEK293 , Replicación Viral , Evasión Inmune , Infecciones por Picornaviridae/inmunología , Infecciones por Picornaviridae/virología , Infecciones por Picornaviridae/metabolismo , Línea CelularRESUMEN
BACKGROUND: Osteoarthritis (OA) is a progressive condition affecting the joints that lacking effective therapy. However, the underlying molecular mechanism has not been fully clarified. METHODS: A model of OA was established in Sprague-Dawley (SD) rats through intra-articularly injected with monoiodoacetate (MIA). Western blot analysis was used to identify the levels of UBE2I and hnRNPA2B1 in articular cartilage. Overexpression and siRNA vectors for UBE2I were constructed and transfected into rat chondrocytes. CCK-8, TUNEL and transwell assay were utilized to assess the cell viability, apoptosis and migration ability. Western blot analysis was used to determine the levels of chondrogenic-specific genes including SOX9, COL2A1, Aggrecan, and PRG4. Then, molecular interactions were confirmed by immunoprecipitation. RESULTS: We observed significant upregulation of UBE2I and hnRNPA2B1 expression in articular cartilage samples of OA. The Pearson correlation analysis revealed positive correlation between UBE2I and hnRNPA2B1 levels. Functional experiments showed that increased UBE2I expression significantly suppressed cell growth, migration, and reduced the expression of chondrogenic-specific genes, while decreasing UBE2I levels had the opposite effects. Molecular interactions between UBE2I and hnRNPA2B1were determined via co-localization and immunoprecipitation. SUMO1 and SUMO3 proteins were enriched by immunoprecipitation using hnRNPA2B1 antibodies. Rescue experiments were performed using SUMOylation inhibitor (2-D08) and SUMOylation activator (N106). Overexpression of UBE2I increased the expression of hnRNPA2B1 in the cytoplasm and decreased the level in the nucleus, which was reversed by the treatment of 2-D08. Conversely, UBE2I knockdown and N106 treatment had the opposite effect. CONCLUSIONS: UBE2I modulated the nuclear translocation of hnRNPA2B1 by promoting SUMOylation in OA.
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Condrocitos , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Osteoartritis , Sumoilación , Enzimas Ubiquitina-Conjugadoras , Animales , Masculino , Ratas , Cartílago Articular/metabolismo , Cartílago Articular/patología , Movimiento Celular , Núcleo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Condrocitos/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Osteoartritis/metabolismo , Osteoartritis/genética , Osteoartritis/patología , Ratas Sprague-Dawley , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
Helicobacter pylori (H. pylori) infection is the primary risk factor for the pathogenesis of gastric cancer (GC). N6-methyladenosine (m6A) plays pivotal roles in mRNA metabolism and hnRNPA2B1 as an m6A reader is shown to exert m6A-dependent mRNA stabilization in cancer. This study aims to explore the role of hnRNPA2B1 in H. pylori-associated GC and its novel molecular mechanism. Multiple datasets and tissue microarray are utilized for assessing hnRNPA2B1 expression in response to H. pylori infection and its clinical prognosis in patients with GC. The roles of hnRNPA2B1 are investigated through a variety of techniques including glucose metabolism analysis, m6A-epitranscriptomic microarray, Ribo-seq, polysome profiling, RIP-seq. In addition, hnRNPA2B1 interaction with poly(A) binding protein cytoplasmic 1 (PABPC1) is validated using mass spectrometry and co-IP. These results show that hnRNPA2B1 is upregulated in GC and correlated with poor prognosis. H. pylori infection induces hnRNPA2B1 upregulation through recruiting NF-κB to its promoter. Intriguingly, cytoplasm-anchored hnRNPA2B1 coordinated PABPC1 to stabilize its relationship with cap-binding eIF4F complex, which facilitated the translation of CIP2A, DLAT and GPX1 independent of m6A modification. In summary, hnRNPA2B1 facilitates the non-m6A translation of epigenetic mRNAs in GC progression by interacting with PABPC1-eIF4F complex and predicts poor prognosis for patients with GC.
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Progresión de la Enfermedad , Infecciones por Helicobacter , Helicobacter pylori , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Proteína I de Unión a Poli(A) , Neoplasias Gástricas , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patología , Humanos , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/metabolismo , Proteína I de Unión a Poli(A)/metabolismo , Proteína I de Unión a Poli(A)/genética , Ratones , Animales , Pronóstico , Modelos Animales de Enfermedad , Masculino , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/genéticaRESUMEN
Although various anti-inflammatory medications, such as ephedrine, are employed to manage cough-variant asthma, their underlying mechanisms are yet to be fully understood. Recent studies suggest that exosomes derived from airway epithelial cells (AECs) contain components like messenger RNAs (mRNAs), micro-RNAs (miRNAs), and long noncoding RNA (lncRNA), which play roles in the occurrence and progression of airway inflammation. This study investigates the influence of AEC-derived exosomes on the efficacy of ephedrine in treating cough-variant asthma. We established a mouse model of asthma and measured airway resistance and serum inflammatory cell levels. Real-time polymerase chain reaction (RT-qPCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA) analyses were used to assess gene and protein expression levels. Exosomes were isolated and characterized. RNA immunoprecipitation (RIP) and RNA pull-down assays were conducted to examine the interaction between hnRNPA2B1 and lnc-TRPM2-AS1. In the ovalbumin (OVA)-challenged mouse model, ephedrine treatment reduced inflammatory responses, airway resistance, and Th1/Th2 cell imbalance. Exosomes from OVA-treated AECs showed elevated levels of lnc-TRPM2-AS1, which were diminished following ephedrine treatment. The exosomal lnc-TRPM2-AS1 mediated the Th1/Th2 imbalance in CD4+ T cells, with its packaging into exosomes being facilitated by hnRNPA2B1. This study unveils a novel mechanism by which ephedrine ameliorates OVA-induced CD4+ T cell imbalance by suppressing AEC-derived exosomal lnc-TRPM2-AS1. These findings could provide a theoretical framework for using ephedrine in asthma treatment.
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Asma , Efedrina , Células Epiteliales , Exosomas , Ratones Endogámicos BALB C , Ovalbúmina , Células Th2 , Animales , Asma/tratamiento farmacológico , Asma/inmunología , Efedrina/farmacología , Exosomas/metabolismo , Ratones , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Th2/inmunología , Células Th2/efectos de los fármacos , Femenino , ARN Largo no Codificante/genética , Humanos , Células TH1/efectos de los fármacos , Células TH1/inmunología , Modelos Animales de EnfermedadRESUMEN
Recent advancements in proteomics technologies using formalin-fixed paraffin-embedded (FFPE) samples have significantly advanced biomarker discovery. Yet, the effects of varying sample preparation protocols on proteomic analyses remain poorly understood. We analyzed mouse liver FFPE samples that varied in fixatives, fixation duration, and storage temperature using LC/MS. We found that variations in fixation duration significantly affected the abundance of specific proteins, showing that HNRNPA2/B1 demonstrated a significant decrease in abundance in samples fixed for long periods, whereas STT3B exhibited a significant increase in abundance in samples fixed for long durations. These findings were supported by immunohistochemical analysis across liver, spleen, and lung tissues, demonstrating a significant decrease in the nuclear staining of HNRNPA2/B1 in long-duration acid formalin(AF)-fixed FFPE samples, and an increase in cytoplasmic staining of STT3B in long-duration neutral buffered formalin-fixed liver and lung tissues and granular staining in all long-duration AF-fixed FFPE tissue types. Similar trends were observed in the long-duration fixed HeLa cells. These results demonstrate that fixation duration critically affects the proteomic integrity of FFPE samples, emphasizing the urgent need for standardized fixation protocols to ensure consistent and reliable proteomic data. SIGNIFICANCE: The quality of FFPE samples is primarily influenced by the fixation and storage conditions. However, previous studies have mainly focused on their impact on nucleic acids and the extent to which different fixation conditions affect changes in proteins has not been evaluated. In addition, to our knowledge, proteomic research focusing on differences in formalin fixation conditions has not yet been conducted. Here, we analyzed FFPE samples with different formalin fixation and storage conditions using LC/MS and evaluated the impact of different fixation conditions on protein variations. Our study unequivocally established formalin fixation duration as a critical determinant of protein variation in FFPE specimens and successfully identified HNRNPA2/B1 and STT3B as potential biomarkers for predicting formalin fixation duration for the first time. The study findings open new avenues for quality assessment in biomedical research and diagnostics.
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Formaldehído , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Proteómica , Fijación del Tejido , Animales , Ratones , Humanos , Proteómica/métodos , Fijación del Tejido/métodos , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Células HeLa , Adhesión en Parafina , Hígado/metabolismo , Hígado/químicaRESUMEN
RNAs, such as noncoding RNA, microRNA, and recently mRNA, have been recognized as signal transduction molecules. CD271, also known as nerve growth factor receptor, has a critical role in cancer, although the precise mechanism is still unclear. Here, we show that CD271 mRNA, but not CD271 protein, facilitates spheroid cell proliferation. We established CD271-/- cells lacking both mRNA and protein of CD271, as well as CD271 protein knockout cells lacking only CD271 protein, from hypopharyngeal and oral squamous cell carcinoma lines. Sphere formation was reduced in CD271-/- cells but not in CD271 protein knockout cells. Mutated CD271 mRNA, which is not translated to a protein, promoted sphere formation. CD271 mRNA bound to hnRNPA2B1 protein at the 3'-UTR region, and the inhibition of this interaction reduced sphere formation. In surgical specimens, the CD271 mRNA/protein expression ratio was higher in the cancerous area than in the noncancerous area. These data suggest CD271 mRNA has dual functions, encompassing protein-coding and noncoding roles, with its noncoding RNA function being predominant in oral and head and neck squamous cell carcinoma.
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Neoplasias de Cabeza y Cuello , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Neoplasias de la Boca , Proteínas del Tejido Nervioso , ARN Mensajero , Receptores de Factor de Crecimiento Nervioso , Carcinoma de Células Escamosas de Cabeza y Cuello , Femenino , Humanos , Masculino , Regiones no Traducidas 3' , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismoRESUMEN
BACKGROUND: Chemoresistance is a major cause of treatment failure in gastric cancer (GC). Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) is an N6-methyladenosine (m6A)-binding protein involved in a variety of cancers. However, whether m6A modification and hnRNPA2B1 play a role in GC chemoresistance is largely unknown. In this study, we aimed to investigate the role of hnRNPA2B1 and the downstream mechanism in GC chemoresistance. METHODS: The expression of hnRNPA2B1 among public datasets were analyzed and validated by quantitative PCR (qPCR), Western blotting, immunofluorescence, and immunohistochemical staining. The biological functions of hnRNPA2B1 in GC chemoresistance were investigated both in vitro and in vivo. RNA sequencing, methylated RNA immunoprecipitation, RNA immunoprecipitation, and RNA stability assay were performed to assess the association between hnRNPA2B1 and the binding RNA. The role of hnRNPA2B1 in maintenance of GC stemness was evaluated by bioinformatic analysis, qPCR, Western blotting, immunofluorescence, and sphere formation assays. The expression patterns of hnRNPA2B1 and downstream regulators in GC specimens from patients who received adjuvant chemotherapy were analyzed by RNAscope and multiplex immunohistochemistry. RESULTS: Elevated expression of hnRNPA2B1 was found in GC cells and tissues, especially in multidrug-resistant (MDR) GC cell lines. The expression of hnRNPA2B1 was associated with poor outcomes of GC patients, especially in those who received 5-fluorouracil treatment. Silencing hnRNPA2B1 effectively sensitized GC cells to chemotherapy by inhibiting cell proliferation and inducing apoptosis both in vitro and in vivo. Mechanically, hnRNPA2B1 interacted with and stabilized long noncoding RNA NEAT1 in an m6A-dependent manner. Furthermore, hnRNPA2B1 and NEAT1 worked together to enhance the stemness properties of GC cells via Wnt/ß-catenin signaling pathway. In clinical specimens from GC patients subjected to chemotherapy, the expression levels of hnRNPA2B1, NEAT1, CD133, and CD44 were markedly elevated in non-responders compared with responders. CONCLUSION: Our findings indicated that hnRNPA2B1 interacts with and stabilizes lncRNA NEAT1, which contribute to the maintenance of stemness property via Wnt/ß-catenin pathway and exacerbate chemoresistance in GC.
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Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Ribonucleoproteínas Nucleares Heterogéneas , ARN Largo no Codificante , Neoplasias Gástricas , Humanos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , ARN Largo no Codificante/genética , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismoRESUMEN
Finding effective therapeutic targets to treat NRAS-mutated melanoma remains a challenge. Long non-coding RNAs (lncRNAs) recently emerged as essential regulators of tumorigenesis. Using a discovery approach combining experimental models and unbiased computational analysis complemented by validation in patient biospecimens, we identified a nuclear-enriched lncRNA (AC004540.4) that is upregulated in NRAS/MAPK-dependent melanoma, and that we named T-RECS. Considering potential innovative treatment strategies, we designed antisense oligonucleotides (ASOs) to target T-RECS. T-RECS ASOs reduced the growth of melanoma cells and induced apoptotic cell death, while having minimal impact on normal primary melanocytes. Mechanistically, treatment with T-RECS ASOs downregulated the activity of pro-survival kinases and reduced the protein stability of hnRNPA2/B1, a pro-oncogenic regulator of MAPK signaling. Using patient- and cell line- derived tumor xenograft mouse models, we demonstrated that systemic treatment with T-RECS ASOs significantly suppressed the growth of melanoma tumors, with no noticeable toxicity. ASO-mediated T-RECS inhibition represents a promising RNA-targeting approach to improve the outcome of MAPK pathway-activated melanoma.
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Melanoma , ARN Largo no Codificante , Humanos , Ratones , Animales , Melanoma/patología , ARN Largo no Codificante/genética , Apoptosis/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , Línea Celular Tumoral , Proteínas de la Membrana/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismoRESUMEN
Although the composition and assembly of stress granules (SGs) are well understood, the molecular mechanisms underlying SG disassembly remain unclear. Here, we identify that heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) is associated with SGs and that its absence specifically enhances the disassembly of arsenite-induced SGs depending on the ubiquitination-proteasome system but not the autophagy pathway. hnRNPA2B1 interacts with many core SG proteins, including G3BP1, G3BP2, USP10, and Caprin-1; USP10 can deubiquitinate G3BP1; and hnRNPA2B1 depletion attenuates the G3BP1-USP10/Caprin-1 interaction but elevates the G3BP1 ubiquitination level under arsenite treatment. Moreover, the disease-causing mutation FUSR521C also disassembles faster from SGs in HNRNPA2B1 mutant cells. Furthermore, knockout of hnRNPA2B1 in mice leads to Sertoli cell-only syndrome (SCOS), causing complete male infertility. Consistent with this, arsenite-induced SGs disassemble faster in Hnrnpa2b1 knockout (KO) mouse Sertoli cells as well. These findings reveal the essential roles of hnRNPA2B1 in regulating SG disassembly and male mouse fertility.
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Arsenitos , Masculino , Animales , Ratones , Arsenitos/toxicidad , ADN Helicasas , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , FertilidadRESUMEN
The lncRNA plays an important role in tumorigenesis and the progression of renal cell carcinoma (RCC). LINC00645 is one of the most different expressed lncRNA between RCC and normal renal tissue. However, the regulatory mechanism of LINC00645 in RCC remains unknown. Our results indicated that LINC00645 inhibited RCC proliferation, migration, and invasion. Mechanistically, HNRNPA2B1 directly bound to ROCK1 mRNA and strengthened its stability. LINC00645 competitively bound to the RRM1 domain, which is responsible for interacting with ROCK1 mRNA, reducing ROCK1 mRNA level by affecting posttranscriptional destabilization. The expression of LINC00645 was significantly reduced in RCC cells, significantly upregulating ROCK1 by abolishing the interaction with HNRNPA2B1, finally promoting RCC proliferation, migration, and invasion. Moreover, RCC cells with lower LINC00645 expression were more sensitive to the ROCK1 inhibitor Y-27632. Our study indicates that decreased expression of LINC00645 promotes the RCC progression via HNRNPA2B1/ROCK1 axis, providing a promising treatment strategy for RCC patients with decreased LINC00645 expression.
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Carcinoma de Células Renales , Neoplasias Renales , Estabilidad del ARN , ARN Largo no Codificante , Quinasas Asociadas a rho , Humanos , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/patología , Quinasas Asociadas a rho/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genéticaRESUMEN
HNRNPA2B1 and HNRNPR stabilize ASCL1 mRNA in neuroblastoma, but whether their regulatory effects depend on m6A modification and whether their function involves ASCL1 remain unknown. This study investigated the m6A-dependent binding of HNRNPA2B1 and HNRNPR to ASCL1 and subsequent regulation, as well as the expression, clinical significance, and function of HNRNPA2B1 and HNRNPR in neuroblastoma. We revealed that METTL14 mediated ASCL1 m6A modification to stabilize ASCL1. HNRNPA2B1 and HNRNPR significantly enriched ASCL1 mRNA by binding to the 5' and 3' untranslated regions, respectively, and METTL14 knockdown reduced this enrichment. Mutations in m6A sites in the untranslated regions of ASCL1 mRNA considerably decreased probe capacity to engage HNRNPA2B1 and HNRNPR. HNRNPR interacts with IGF2BP1, and knocking down either impaired binding to ASCL1 mRNA. HNRNPA2B1 and HNRNPR knockdown suppressed neuroblastoma cell growth and invasion, while ASCL1 overexpression restored these effects. The high HNRNPA2B1 and HNRNPR expression in neuroblastoma correlated with ASCL1 expression. Thus, HNRNPA2B1 and HNRNPR bind and stabilize ASCL1 mRNA in an m6A-dependent manner to promote neuroblastoma progression. This study not only discovered a new mechanism underlying the high ASCL1 expression in neuroblastoma but also identified the HNRNPA2B1/HNRNPR/ASCL1 axis as a promising target for inhibiting neuroblastoma progression.
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Adenina/análogos & derivados , Neuroblastoma , Humanos , Neuroblastoma/genética , Regiones no Traducidas 3' , ARN Mensajero/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ribonucleoproteínas Nucleares HeterogéneasRESUMEN
Oral squamous cell carcinoma (OSCC), as a common type of oral malignancy, has an unclear pathogenesis. N6 methyladenosine (m6A) is a reversible and dynamic process that participates in the modulation of cancer pathogenesis and development. As an m6A recognition protein (reader), heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1) show abnormally high expression in cancers. Forkhead box Q1 (FOXQ1), an oncogenic transcription factor, controls multiple biological processes (e.g., embryonic development, cell differentiation, and apoptosis, impacting the initiation and progression of cancers by mediating signaling pathways together with epithelial-mesenchymal transition). Through the Cancer Genome Atlas database screening along with clinical and laboratory experiments, in head and neck squamous cell carcinoma, we found a correlation between HNRNPA2B1 and FOXQ1 gene expression, with shared m6A motifs between HNRNPA2B1 and FOXQ1 mRNA sequences. Silencing or overexpression of HNRNPA2B1 in OSCC cells affected the malignant phenotypes of OSCC cells in vitro, and depletion of HNRNPA2B1 retarded tumor growth in vivo. HNRNPA2B1 could bind to m6A-modified FOXQ1 mRNA to enhance its mRNA stability, resulting in up-regulation of FOXQ1 protein expression. To conclude, HNRNPA2B1 was upregulated in OSCC and enhanced OSCC cell malignant phenotypes by stabilizing m6A-modified FOXQ1 mRNA, eventually aggravating the malignancy and tumorigenicity of OSCC. This study accelerates the recognition of the potency of m6A modification in OSCC and paves the path for OSCC's targeted diagnosis and therapy.
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Factores de Transcripción Forkhead , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Neoplasias de la Boca , Estabilidad del ARN , Humanos , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Estabilidad del ARN/genética , Animales , Ratones , Ratones Desnudos , Proliferación Celular , Adenosina/metabolismo , Adenosina/análogos & derivados , Adenosina/genética , Carcinogénesis/genética , Línea Celular Tumoral , ARN Mensajero/genética , ARN Mensajero/metabolismo , Femenino , Ensayos Antitumor por Modelo de Xenoinjerto , Masculino , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Ratones Endogámicos BALB C , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismoRESUMEN
Galectin-3, well characterized as a glycan binding protein, has been identified as a putative RNA binding protein, possibly through participation in pre-mRNA maturation through interactions with splicosomes. Given recent developments with cell surface RNA biology, the putative dual-function nature of galectin-3 evokes a possible non-classical connection between glycobiology and RNA biology. However, with limited functional evidence of a direct RNA interaction, many molecular-level observations rely on affinity reagents and lack appropriate genetic controls. Thus, evidence of a direct interaction remains elusive. We demonstrate that antibodies raised to endogenous human galectin-3 can isolate RNA-protein crosslinks, but this activity remains insensitive to LGALS3 knock-out. Proteomic characterization of anti-galectin-3 IPs revealed enrichment of galectin-3, but high abundance of hnRNPA2B1, an abundant, well-characterized RNA-binding protein with weak homology to the N-terminal domain of galectin-3, in the isolate. Genetic ablation of HNRNPA2B1, but not LGALS3, eliminates the ability of the anti-galectin-3 antibodies to isolate RNA-protein crosslinks, implying either an indirect interaction or cross-reactivity. To address this, we introduced an epitope tag to the endogenous C-terminal locus of LGALS3. Isolation of the tagged galectin-3 failed to reveal any RNA-protein crosslinks. This result suggests that the galectin-3 does not directly interact with RNA and may be misidentified as an RNA-binding protein, at least in HeLa where the putative RNA associations were first identified. We encourage further investigation of this phenomenon employ gene deletions and, when possible, endogenous epitope tags to achieve the specificity required to evaluate potential interactions.
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Galectina 3 , ARN , Humanos , Epítopos , Galectina 3/genética , Galectina 3/metabolismo , Galectinas/metabolismo , Proteómica , Proteínas de Unión al ARNRESUMEN
BACKGROUND: Dysregulation of cholesterol metabolism is a significant characteristic of glioma, yet the underlying mechanisms are largely unknown. N6-methyladenosine (m6A) modification has been implicated in promoting tumor development and progression. The aim of this study was to determine the key m6A regulatory proteins involved in the progression of glioma, which is potentially associated with the reprogramming of cholesterol homeostasis. METHODS: Bioinformatics analysis was performed to determine the association of m6A modification with glioma malignancy from The Cancer Genome Atlas and Genotype-Tissue Expression datasets. Glioma stem cell (GSC) self-renewal was determined by tumor sphere formation and bioluminescence image assay. RNA sequencing and lipidomic analysis were performed for cholesterol homeostasis analysis. RNA immunoprecipitation and luciferase reporter assay were performed to determine hnRNPA2B1-dependent regulation of sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR) mRNA. The methylation status of hnRNPA2B1 promoter was determined by bioinformatic analysis and methylation-specific PCR assay. RESULTS: Among the m6A-regulatory proteins, hnRNPA2B1 was demonstrated the most important independent prognostic risk factor for glioma. hnRNPA2B1 ablation exhibited a significant tumor-suppressive effect on glioma cell proliferation, GSC self-renewal and tumorigenesis. hnRNPA2B1 triggers de novo cholesterol synthesis by inducing HMGCR through the stabilization of SREBP2 mRNA. m6A modification of SREBP2 or LDLR mRNA is required for hnRNPA2B1-mediated mRNA stability. The hypomethylation of cg21815882 site on hnRNPA2B1 promoter confers elevated expression of hnRNPA2B1 in glioma tissues. The combination of targeting hnRNPA2B1 and cholesterol metabolism exhibited remarkable antitumor effects, suggesting valuable clinical implications for glioma treatment. CONCLUSIONS: hnRNPA2B1 facilitates cholesterol uptake and de novo synthesis, thereby contributing to glioma stemness and malignancy.
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Colesterol , Glioma , Humanos , Colesterol/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Glioma/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , HomeostasisRESUMEN
LINC00355 is involved in the tumorigenesis of several types of cancer. We verified that LINC00355 is upregulated in gastric cancer (GC) and contributes to GC cells' proliferation and metastasis. RNA sequencing (RNA-seq) and rescue assays suggested that LINC00355 controls gastric carcinogenesis by regulating the expression of cell division cycle 42 (CDC42) guanosine triphosphatase (GTPases), thereby activating their downstream pathways. Most previous studies have shown that LINC00355 acts as a ceRNA by sponging miRNAs to modulate downstream gene expression. Our group focus on epigenetic regulatory potential of LINC00355 in gene expression. Mechanistically, LINC00355 binds to p300 histone acetyltransferase, specifying the histone modification pattern on the CDC42 promoter to activate CDC42 transcription, thereby altering GC cell biology. In addition, HNRNPA2B1, which is upregulated by LINC00355, recognizes the N6-methyladenosine (m6A) sites of CDC42 and enhances the stability of CDC42 mRNA transcripts. Therefore, LINC00355 is mechanistically, functionally, and clinically oncogenic in GC cells.
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Adenina/análogos & derivados , MicroARNs , Neoplasias Gástricas , Humanos , ARN Mensajero/genética , Carcinogénesis/genética , Transformación Celular Neoplásica , Neoplasias Gástricas/patologíaRESUMEN
INTRODUCTION: Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator mediating adaptive responses to hypoxia. It is up-regulated in the tumor microenvironment and recognized as an effective anticancer drug target. Previously, we discovered that the natural compound moracin-O and its synthetic derivative MO-460 inhibited HIF-1α via hnRNPA2B1. OBJECTIVES: This study aimed to develop novel HIF-1 inhibitors for cancer chemotherapy by harnessing the potential of the natural products moracins-O and P. METHODS: In an ongoing search for novel HIF-1 inhibitors, a series of nature-inspired benzofurans with modifications on the chiral rings of moracins-O and P were synthesized. They showed improved chemical tractability and were evaluated for their inhibitory activity on HIF-1α accumulation under hypoxic conditions in HeLa CCL2 cells. The most potent derivative's chemical-based toxicities, binding affinities, and in vivo anti-tumorigenic effects were evaluated. Further, we examined whether our compound, MO-2097, exhibited anticancer effects in three-dimensional cultured organoids. RESULTS: Herein, we identified a novel synthetic chiral-free compound, MO-2097, with reduced structural complexity and increased efficiency. MO-2097 exhibited inhibitory effects on hypoxia-induced HIF-1α accumulation in HeLa CCL2 cells via inhibition of hnRNPA2B1 protein, whose binding affinities were confirmed by isothermal titration calorimetry analysis. In addition, MO-2097 demonstrated in vivo efficacy and biocompatibility in a BALB/c mice xenograft model. The immunohistochemistry staining of MO-2097-treated tissues showed decreased expression of HIF-1α and increased levels of apoptosis marker cleaved caspase 3, confirming in vivo efficacy. Furthermore, we confirmed that MO-2097 works effectively in cancer patient-based organoid models. CONCLUSION: MO-2097 represents a promising new generation of chemotherapeutic agents targeting HIF-1α inhibition via hnRNPA2B1, requiring further investigation.