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Polyamines act as protective compounds directly protecting plants from stress-related damage, while also acting as signaling molecules to participate in serious abiotic stresses. However, the molecular mechanisms underlying these effects are poorly understood. Here, we utilized metabolome genome-wide association study to investigate the polyamine content of wild and cultivated tomato accessions, and we discovered a new gene cluster that drove polyamine content during tomato domestication. The gene cluster contains two polyphenol oxidases (SlPPOE and SlPPOF), two BAHD acyltransferases (SlAT4 and SlAT5), a coumaroyl-CoA ligase (Sl4CL6), and a polyamine uptake transporter (SlPUT3). SlPUT3 mediates polyamine uptake and transport, while the five other genes are involved in polyamine modification. Further salt tolerance assays demonstrated that SlPPOE, SlPPOF, and SlAT5 overexpression lines showed greater phenolamide accumulation and salt tolerance as compared with wild-type (WT). Meanwhile, the exogenous application of Spm to SlPUT3-OE lines displayed salt tolerance compared with WT, while having the opposite effect in slput3 lines, confirms that the polyamine and phenolamide can play a protective role by alleviating cell damage. SlPUT3 interacted with SlPIP2;4, a H2O2 transport protein, to maintain H2O2 homeostasis. Polyamine-derived H2O2 linked Spm to stress responses, suggesting that Spm signaling activates stress response pathways. Collectively, our finding reveals that the H2O2-polyamine-phenolamide module coordinately enhanced tomato salt stress tolerance and provide a foundation for tomato stress-resistance breeding.
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The methyltransferase complex (MTC) deposits N6-adenosine (m6A) onto RNA, whereas the microprocessor produces microRNA. Whether and how these two distinct complexes cross-regulate each other has been poorly studied. Here we report that the MTC subunit B tends to form insoluble condensates with poor activity, with its level monitored by the 20S proteasome. Conversely, the microprocessor component SERRATE (SE) forms liquid-like condensates, which in turn promote the solubility and stability of the MTC subunit B, leading to increased MTC activity. Consistently, the hypomorphic lines expressing SE variants, defective in MTC interaction or liquid-like phase behaviour, exhibit reduced m6A levels. Reciprocally, MTC can recruit the microprocessor to the MIRNA loci, prompting co-transcriptional cleavage of primary miRNA substrates. Additionally, primary miRNA substrates carrying m6A modifications at their single-stranded basal regions are enriched by m6A readers, which retain the microprocessor in the nucleoplasm for continuing processing. This reveals an unappreciated mechanism of phase separation in RNA modification and processing through MTC and microprocessor coordination.
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Methyltransferase complex (MTC) deposits N 6-adenosine (m 6 A) onto RNA, whereas microprocessor produces miRNA. Whether and how these two distinct complexes cross-regulate each other has been poorly studied. Here we report that the MTC subunit B (MTB) tends to form insoluble condensates with poor activity, with its level monitored by 20S proteasome. Conversely, the microprocessor component SERRATE (SE) forms liquid-like condensates, which in turn promotes solubility and stability of MTB, leading to increased MTC activity. Consistently, the hypomorphic lines expressing SE variants, defective in MTC interaction or liquid-like phase behavior, exhibit reduced m 6 A level. Reciprocally, MTC can recruit microprocessor to MIRNA loci, prompting co-transcriptional cleavage of primary miRNA (pri-miRNAs) substrates. Additionally, pri-miRNAs carrying m 6 A modifications at their single-stranded basal regions are enriched by m 6 A readers, which retain microprocessor in the nucleoplasm for continuing processing. This reveals an unappreciated mechanism of phase separation in RNA modification and processing through MTC and microprocessor coordination.
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NUDT15 encodes nucleotide triphosphate diphosphatase that is responsible for metabolizing purine analog drugs, and its genetic mutation results in severe side effects from thiopurine therapy. However, the functions of Nudt15 in leukemic stem cells (LSCs) and hematopoietic stem cells (HSCs) remain unknown. Here we reveal the Nudt15-regulating self-renewal of both mouse LSCs and HSCs. Our data show that Nudt15 negatively regulates murine leukemogenesis and its deficiency prolongs the survival of murine AML recipients by impairing LSC self-renewal, while Nudt15 ablation markedly enhances mouse HSC regenerative potential and self-renewal. Mechanistically, Nudt15 modulates inflammatory signaling in mouse LSCs and HSCs, leading to divergent self-renewal outcomes. Nudt15 depletion inhibits mouse LSC self-renewal by downregulating Ifi30, resulting in elevating intracellular ROS level. Gata2, a key regulator, is required for Nudt15-mediating inflammatory signaling in mouse HSCs. Collectively, our results present new crucial roles of Nudt15 in maintaining the functions of mouse LSC and HSC through inflammatory signaling and have a new insight into clinical implications.
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Hematopoese , Células-Tronco Hematopoéticas , Pirofosfatases , Transdução de Sinais , Animais , Humanos , Camundongos , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA2/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Inflamação/metabolismo , Inflamação/patologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/etiologia , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Pirofosfatases/genética , Pirofosfatases/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer, and the early detection and diagnosis of this disease are crucial in reducing mortality rates. The timely diagnosis of LUAD is essential for controlling tumour development and enabling early surgical treatment. GPR56 is a vital G protein-coupled receptor and its role in T lymphocytes has received considerable attention. However, its function in B cells remains unclear. This study aimed to investigate the significance of GPR56 in LUAD. We found that GPR56 exhibited a significant increase in circulating plasmablasts and a decrease in new memory B cells. GPR56 expression in B cells was significantly reduced after LPS stimulation and the proportion of HLA-DR+ and CD40+ proportions were also decreased in GPR56+ B cells after stimulation. Additionally, GPR56 exhibited significant down-regulation in circulating B cell subsets of early-stage LUAD patients, and there were significant correlations between GPR56+ B cell subsets and tumour markers. In conclusion, GPR56 could reflect the hypoactivation state of B cells and the decreased proportion of GPR56+ B cell subset in LUAD patients can signify the active humoral immunity in vivo. The expression of GPR56 in B cells could potentially hold value in the early diagnosis of LUAD.
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Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Feminino , Idoso , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Ativação Linfocitária , Regulação para Baixo , Estadiamento de Neoplasias , Imunidade Humoral , Biomarcadores Tumorais/metabolismoRESUMO
This study aims to elucidate the expression and functionality of SIT1 in circulating CD8/CD4 + T cells in humans and to delineate its significance in systemic lupus erythematosus (SLE) patients. We employed multiparametric flow cytometry to investigate the expression of SIT1 in circulating CD8/CD4 + T cells and their respective subsets, comparing healthy controls (HCs) with SLE patients. Furthermore, we assessed the levels of granzyme B, perforin, IL-17, and IFN-γ in SIT1-related CD8/CD4 + T cells from both HCs and SLE patients, both before and after PMA stimulation. Clinically, we conducted receiver operating characteristic curve analysis and correlation analysis to evaluate the clinical relevance of SIT1-related CD8/CD4 + T cells in SLE patients. SIT1 exhibited higher expression in CD4 + T cells, with SIT1 - T cells demonstrating elevated levels of granzyme B, perforin, and IFN-γ compared to SIT1 + T cells. PMA-stimulated T cells exhibited reduced SIT1 expression compared to unstimulated T cells. SLE patients displayed increased SIT1 + proportions in CD8 + T cells and decreased SIT1 + CD4 + T cell numbers. Additionally, SIT1 + cells in SLE patients exhibited significantly higher levels of granzyme B and perforin compared to HCs. SIT1 + cells demonstrated significant associations with clinical indicators in SLE patients, with indicators related to SIT1 proving valuable in the diagnosis of SLE patients. SIT1 is inversely correlated with T cell activation. In SLE patients, SIT1 expression is altered in T cells concomitant with an augmented secretion of cytotoxic molecules. This upregulation may contribute to the pathogenesis of SLE and enhance its diagnostic potential.
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Proteínas Adaptadoras de Transdução de Sinal , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Granzimas , Lúpus Eritematoso Sistêmico , Glicoproteínas de Membrana , Perforina , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citotoxicidade Imunológica , Citometria de Fluxo , Granzimas/metabolismo , Interferon gama/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/metabolismo , Ativação Linfocitária/imunologia , Perforina/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
OBJECTIVE: This study aimed to investigate the expression of GPR56 in the T cells of early-stage lung adenocarcinoma (LUAD) patients and clarify its diagnostic significance. METHODS: Blood samples were collected from 32 patients with stage IA LUAD and 31 healthy controls. GPR56 and perforin were analysed in circulating T-cell subsets by flow cytometry. In addition, a correlation between perforin and GPR56 expression was detected. Changes in GPR56+ cells in early LUAD patients were analysed, and the diagnostic significance of GPR56+ T cells for early LUAD was studied by receiver operating characteristic (ROC) curve analysis. RESULTS: The expression of GPR56 in CD8+ T cells from early-stage LUAD patients was significantly greater than that in CD4+ T cells. The percentage of perforin-positive GPR56+ cells in early-stage LUAD patients was high. GPR56 levels in the T cells of LUAD patients were significantly lower than those in healthy controls. ROC analysis revealed that the area under the curve for the percentage of GPR56-positive CD8+ TEMRA cells to distinguish early-stage LUAD patients from healthy individuals- reached 0.7978. CONCLUSION: The decreased expression of GPR56 in the peripheral blood of early-stage LUAD patients correlated with perforin levels, reflecting compromised antitumor immunity and aiding early-stage LUAD screening.
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Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Estadiamento de Neoplasias , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Masculino , Feminino , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/diagnóstico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Pessoa de Meia-Idade , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Idoso , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Perforina/metabolismo , Perforina/genética , Curva ROC , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Biomarcadores Tumorais/metabolismo , AdultoRESUMO
Leukemia stem cells (LSC) represent a crucial and rare subset of cells present in acute myeloid leukemia (AML); they play a pivotal role in the initiation, maintenance, and relapse of this disease. Targeting LSC holds great promise for preventing AML relapse and improving long-term outcomes. However the precise molecular mechanisms governing LSC self-renewal are still poorly understood. Here, we present compelling evidence that the expression of miR-30e-5p, a potential tumor-suppressive microRNA, is significantly lower in AML samples than in healthy bone marrow samples. Forced expression of miR- 30e effectively inhibits leukemogenesis, impairs LSC self-renewal, and delays leukemia progression. Mechanistically, Cyb561 acts as a direct target of miR-30e-5p in LSC, and its deficiency restricts the self-renewal of LSC by activating reactive oxygen series signaling and markedly prolongs recipients' survival. Moreover, genetic or pharmacological overexpression of miR-30e-5p or knockdown of Cyb561 suppresses the growth of human AML cells. In conclusion, our findings establish the crucial role of the miR-30e-5p/Cyb561/ROS axis in finely regulating LSC self-renewal, highlighting Cyb561 as a potential therapeutic target for LSC-directed therapies.
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Leucemia Mieloide Aguda , MicroRNAs , Humanos , Espécies Reativas de Oxigênio , Autorrenovação Celular/genética , MicroRNAs/genética , Transdução de Sinais , Recidiva , Proliferação de Células/genética , Linhagem Celular TumoralRESUMO
OBJECTIVE: This study investigated CX3CR1 expression in human peripheral blood T lymphocytes and their subsets, exploring changes in SLE patients and its diagnostic potential. METHODS: Peripheral blood samples from 31 healthy controls and 50 SLE patients were collected. RNA-Seq data from SLE patient PBMCs were used to analyze CX3CR1 expression in T cells. Flow cytometry determined CX3CR1-expressing T lymphocyte subset proportions in SLE patients and healthy controls. Subset composition and presence of GZMB, GPR56, and perforin in CX3CR1+ T lymphocytes were analyzed. T cell-clinical indicator correlations were assessed. ROC curves explored CX3CR1's diagnostic potential for SLE. RESULTS: CX3CR1+CD8+ T cells exhibited higher GPR56, perforin, and GZMB expression than other T cell subsets. The proportion of CX3CR1+ was higher in TEMRA and lower in Tn and TCM. PMA activation reduced CX3CR1+ T cell proportions. Both RNA-Seq and flow cytometry revealed elevated CX3CR1+ T cell proportions in SLE patients. Significantly lower perforin+ and GPR56+ proportions were observed in CX3CR1+CD8+ T cells in SLE patients. CX3CR1+ T cells correlated with clinical indicators. CONCLUSION: CX3CR1+ T cells display cytotoxic features, with heightened expression in CD8+ T cells, particularly in adult SLE patients. Increased CX3CR1 expression in SLE patient T cells suggests its potential as an adjunctive diagnostic marker for SLE.
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Antineoplásicos , Lúpus Eritematoso Sistêmico , Adulto , Humanos , Perforina/genética , Perforina/metabolismo , Regulação para Cima , Subpopulações de Linfócitos T , Linfócitos T CD8-Positivos , Antineoplásicos/metabolismo , Citometria de Fluxo , Receptor 1 de Quimiocina CX3C/metabolismoRESUMO
Cytarabine-resistant acute myeloid leukemia (AML) is a common phenomenon, necessitating the search for new chemotherapeutics. WEE1 participates in cell cycle checkpoint signaling and inhibitors targeting WEE1 (WEE1i) constitute a potential novel strategy for AML treatment. HDAC (histone deacetylase) inhibitors have been shown to enhance the anti-tumor effects of WEE1i but molecular mechanisms of HDAC remain poorly characterized. In this study, the WEE1 inhibitor PD0166285 showed a relatively good anti-leukemia effect. Notably, PD0166285 can arise the expression of HDAC11 which was negatively correlated with survival of AML patients. Moreover, HDAC11 can reduced the anti-tumor effect of PD0166285 through an effect on p53 stability and the changes in phosphorylation levels of MAPK pathways. Overall, the cell cycle inhibitor, PD0166285, is a potential chemotherapeutic drug for AML. These fundings contribute to a functional understanding of HDAC11 in AML.
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Proteínas de Ciclo Celular , Leucemia Mieloide Aguda , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Ubiquitina/farmacologia , Ubiquitina/uso terapêutico , Proteínas Nucleares/metabolismo , Leucemia Mieloide Aguda/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Apoptose , Linhagem Celular TumoralRESUMO
OBJECTIVE: This study aimed to investigate the changes and significance of circulating Helios-associated T cell subsets in patients with early-stage lung adenocarcinoma (LUAD). METHODS: Blood samples were collected from 35 healthy controls and 34 patients with early-stage LUAD. Flow cytometry was used to analyze various CD4+ T cell subsets, including regulatory T(Treg) cells, follicular regulatory T(Tfr) cells, follicular helper T (Tfh) cells, and conventional T (con-T) cells. Correlation analysis was conducted to investigate the association of Helios-related subsets with clinical indicators. The ROC curve was used to explore the potential clinical value of Helios+ T cell subsets in the screening of patients with early LUAD. Fifteen of these patients were tracked after lung cancer resection and changes in Helios+ T cell subsets before and after treatment were analyzed. RESULTS: The percentage and absolute number of Tregs were up-regulated in LUAD patients while Tfh and con-T cells expressing Helios were down-regulated. Absolute counts of Tfr and con-T cells and Helios expression in Tfr and Treg decreased significantly after resection. Helios+ Tfh and con-T were negatively correlated with certain tumor markers. Areas under the curve (AUCs) of percentages and absolute counts of Helios+ Tfh, Treg, Tfr and con-T cells to distinguish early LUAD from healthy individuals were 0.7277, 0.5697, 0.5718, 0.7210 (percentages), 0.7336, 0.7378, 0.5908 and 0.7445(absolute numbers), respectively. CONCLUSION: Helios+ T cell subsets in peripheral blood of early-stage LUAD patients has changed significantly, which may be related to the pathogenesis of LUAD and could help for early diagnosis of LUAD.
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Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Detecção Precoce de Câncer , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Adenocarcinoma de Pulmão/diagnóstico , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Linfócitos T Auxiliares-Indutores/metabolismo , Fatores de Transcrição Forkhead/metabolismoRESUMO
Chinese herbal medicine and Chinese patent medicine have been widely applied for cancer care in China. Rupestonic acid, an active ingredient of Artemisia rupestris L., has recently been confirmed to have certain anti-tumor effects in vitro. In this study, we employed the application of a commonly devoted triphenylamine as a fluorophore and the addition of 2,4-thiazolidinedione as a bridge to integrate rupestonic acid into the AIE system to create an fluorescent probe with anti-tumor properties. The spectral, cytotoxic, and cellular imaging properties of the probe were measured. Its promising responses make possible the application of the probe in antitumor theragnostic systems.
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BACKGROUND: miR-454-3p is considered to have a crucial role in cancer progression, but the potential involvement in acute myeloid leukemia (AML) remains unclear. METHODS: Expression of miR-454-3p and ZEB2 mRNA and protein were quantified in AML cell lines. Cells were transfected with miR-454-3p inhibitor or mimic and cell growth was assessed by colony formation and CCK-8 assays and the cell cycle, apoptosis and autophagy were investigated by Western blotting, flow cytometry, immunofluorescence and 3-methyladenine (3-MA) treatment. RESULTS: miR-454-3p expression was attenuated in AML cells. miR-454-3p overexpression attenuated cell growth and stimulated cell cycle arrest, apoptosis and autophagy. Dual-luciferase reporter assays and bioinformatics analysis showed that AML progression was inhibited when miR-454-3p regulated ZEB2, an effect confirmed by rescue assays. 3-MA reduced the autophagy-inducing effect of ZEB2 knockdown and indicated that autophagy induced apoptosis. miR-454-3p downregulated p-mTOR/p-AKT levels in AML cells. CONCLUSION: The novel role of miR-454-3p as a tumor inhibitor in AML via regulation of the ZEB2/AKT/mTOR axis was demonstrated, indicating miR-454-3p as a potential new molecular target for AML.
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Leucemia Mieloide Aguda , MicroRNAs , Humanos , Proteínas Proto-Oncogênicas c-akt , Apoptose , Autofagia/genética , Leucemia Mieloide Aguda/genética , Serina-Treonina Quinases TOR , MicroRNAs/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genéticaRESUMO
Developing a novel tool capable of real-time monitoring and simultaneously quantifying of both intra/extracellular chemical signals across the large-scale brain is the key bottleneck for understanding the interactions between the molecules inside and outside neurons. Here we built up a high-density intra/extracellular optophysiology platform, together with developing two probes for specific recognition of L-cysteine (Cys) and dopamine (DA), for simultaneously quantifying of both intracellular Cys and extracellular DA with high selectivity and accuracy across the brain of freely moving animals, as well as recording electrical signals. Using this powerful tool, it was found that intracellular Cys regulated extracellular DA through inducing the expression of tyrosine hydroxylase in the depressed mice brain. We also established the functional networks of Cys and DA across 32 brain regions in freely moving animals. More importantly, it was discovered that depression reduced the correlations between adjacent brain regions, which was recovered by the treatment of N-acetyl-l-cysteine.
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Encéfalo , Dopamina , Camundongos , Animais , Encéfalo/metabolismo , Dopamina/química , Acetilcisteína , Neurônios/metabolismoRESUMO
BACKGROUND: Polo-like kinase 4 (PLK4) is a crucial regulator for centriole replication and is reported to be aberrantly expressed in various cancers, where it participates to tumorigenesis. However, PLK4 effect in acute myeloid leukemia (AML), is still uncertain. This study investigates the function of PLK4 in AML. METHODS: Quantitative real-time PCR was used to measure the level of PLK4. Centrinone, a selective PLK4 small molecule inhibitor, was used for PLK4 inhibition and explore its effect in AML cells. The cell growth was detected by the CCK8, while the cell cycle and apoptosis were assessed by flow cytometry. The level of proteins associated with apoptosis, cell cycle and endoplasmic reticulum (ER) stress were analyzed by western blotting. RESULTS: PLK4 was overexpressed in AML cells. PLK4 knockdown or its specific inhibition by centrinone induced G2/M phase arrest via suppressing the expression of cyclin B1 and Cdc2 and promoting the level of proapoptotic proteins. Moreover, PLK4 targeting enhanced the level of proteins related to ER stress, such as GRP78, ATF4, ATF6, and CHOP. CONCLUSION: These findings demonstrated that targeting PLK4 can induce apoptosis, G2/M and ER stress in AML cells.
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Apoptose , Leucemia Mieloide Aguda , Humanos , Regulação para Baixo , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular , Leucemia Mieloide Aguda/genética , Proteínas Serina-Treonina Quinases/genéticaRESUMO
BACKGROUND: Studies have confirmed that acute myeloid leukemia (AML) cells with DNA methyltransferase 3A Arg882His (DNMT3A R882H) mutation show an increased proliferation capability. However, the associated mechanism is still unclear. Glycolysis is involved in regulating malignant proliferation of cancer cell. Hence, we analyzed whether the DNMT3A R882H mutation interferes with glycolysis and thereby influences AML cell proliferation. METHODS: We generated AML cell line carrying a DNMT3A-R882H mutation and compared it with the wild type (DNMT3A-WT) with regard to glycolysis regulation. Moreover, we analyzed the cell line's proliferation and apoptosis by a CCK-8 assay, western blotting, and flow cytometry. The role of NRF2/NQO1 signaling in regulating glycolysis was investigated by NRF2-knockdown and Brusatol (specific inhibitor of NRF2) treatment. RESULTS: DNMT3A R882H cells had a higher glucose transport capacity compared to WT cells and their viability could be reduced by glucose deprivation. Moreover, daunorubicin had a slight inhibitory effect on glycolysis while glycolysis inhibition re-sensitized mutant cells to daunorubicin. Obviously, DNMT3A R882H mutation activated the NRF2/NQO1 pathway and enhanced the glycolytic activity in mutant cells. CONCLUSION: Taken together, these results suggest a novel mechanism by which a DNMT3A R882H mutation promotes glycolysis via activation of NRF2/NQO1 pathway. A parallel glycolysis inhibition adds to the anticancer effects of daunorubicin which might lead to a novel therapeutic approach for the treatment of AML patients carrying a DNMT3A R882H mutation.
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DNA Metiltransferase 3A , Leucemia Mieloide Aguda , Humanos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Sobrevivência Celular , Metilação de DNA , Mutação/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Daunorrubicina/farmacologia , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismoRESUMO
BACKGROUND: DNA methyltransferase 3A (DNMT3A) often mutate on arginine 882 (DNMT3AR882) in acute myeloid leukemia (AML). AML patients with DNMT3A R882 mutation are usually resistant to daunorubicin treatment; however, the associated mechanism is still unclear. Therefore, it is urgent to investigate daunorubicin resistance in AML patients with DNMT3A R882 mutant. METHOD: AML cell lines with DNMT3A-wild type (DNMT3A-WT), and DNMT3A-Arg882His (DNMT3A-R882H) mutation were constructed to investigate the role of DNMT3A R882H mutation on cell proliferation, apoptosis and cells' sensitivity to Danunorubin. Bioinformatics was used to analyze the role of nuclear factor-E2-related factor (NRF2) in AML patients with DNMT3A R882 mutation. The regulatory mechanism of DNMT3A R882H mutation on NRF2 was studied by Bisulfite Sequencing and CO-IP. NRF2 inhibitor Brusatol (Bru) was used to explore the role of NRF2 in AML cells carried DNMT3A R882H mutation. RESULTS: AML cells with a DNMT3A R882H mutation showed high proliferative and anti-apoptotic activities. In addition, mutant cells were less sensitive to daunorubicin and had a higher NRF2 expression compared with those in WT cells. Furthermore, the NRF2/NQO1 pathway was activated in mutant cells in response to daunorubicin treatment. DNMT3A R882H mutation regulated the expression of NRF2 via influencing protein stability rather than decreasing methylation of NRF2 promoter. Also, NRF2/NQO1 pathway inhibition improved mutant cells' sensitivity to daunorubicin significantly. CONCLUSION: Our findings identified NRF2 as an important player in the regulation of cell apoptosis through which helps mediate chemoresistance to daunorubicin in AML cells with DNMT3A R882H mutation. Targeting NRF2 might be a novel therapeutic approach to treat AML patients with a DNMT3A R882H mutation. Video abstract.
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DNA (Citosina-5-)-Metiltransferases , Leucemia Mieloide Aguda , NAD(P)H Desidrogenase (Quinona) , Fator 2 Relacionado a NF-E2 , Humanos , Daunorrubicina/farmacologia , Daunorrubicina/uso terapêutico , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação/genética , NAD(P)H Desidrogenase (Quinona)/genética , Fator 2 Relacionado a NF-E2/genética , Resistencia a Medicamentos AntineoplásicosRESUMO
MicroRNA (miRNA)-222-3p is overexpressed in numerous tumors, where it acts as an oncogene. Although miRNA-222 is highly expressed in acute myeloid leukemia (AML), its functions and the mechanisms underlying these functions have not yet been fully elucidated. This study aimed to investigate the regulatory roles of miRNA-222-3p in AML and the molecular mechanisms underlying these roles. In this study, we observed that miRNA-222-3p increased the viability and suppressed the apoptosis of AML cells. Axin2 was demonstrated to be a direct target of miRNA-222-3p, which when overexpressed, inhibited Axin2 expression and stimulated the Wnt/ß-catenin pathway. In contrast, upregulation of Axin2 expression levels reduced the viability and enhanced the apoptosis of AML cells. Moreover, it partially reversed the effects of the miRNA-222-3p mimic on the proliferation and apoptosis of, and modulation of the Wnt/ß-catenin pathway in, AML cells. Taken together, this study provides strong evidence that miRNA-222-3p can serve as a molecular target for AML treatment.
Assuntos
Proteína Axina , Proliferação de Células , Leucemia Mieloide Aguda , MicroRNAs , Apoptose , Proteína Axina/genética , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , MicroRNAs/metabolismo , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMO
African swine fever (ASF), an acute, severe, highly contagious disease caused by African swine fever virus (ASFV) infection in domestic pigs and boars, has a mortality rate of up to 100%. Because effective vaccines and treatments for ASF are lacking, effective control of the spread of ASF remains a great challenge for the pig industry. Host epigenetic regulation is essential for the viral gene transcription. Bromodomain and extraterminal (BET) family proteins, including BRD2, BRD3, BRD4, and BRDT, are epigenetic "readers" critical for gene transcription regulation. Among these proteins, BRD4 recognizes acetylated histones via its two bromodomains (BD1 and BD2) and recruits transcription factors, thereby playing a pivotal role in transcriptional regulation and chromatin remodeling during viral infection. However, how BET/BRD4 regulates ASFV replication and gene transcription is unknown. Here, we randomly selected 12 representative BET family inhibitors and compared their effects on ASFV infection in pig primary alveolar macrophages (PAMs). These were found to inhibit viral infection by interfering viral replication. The four most effective inhibitors (ARV-825, ZL0580, I-BET-762, and PLX51107) were selected for further antiviral activity analysis. These BET/BRD4 inhibitors dose dependently decreased the ASFV titer, viral RNA transcription, and protein production in PAMs. Collectively, we report novel function of BET/BRD4 inhibitors in inducing suppression of ASFV infection, providing insights into the role of BET/BRD4 in the epigenetic regulation of ASFV and potential new strategies for ASF prevention and control. IMPORTANCE Due to the continuing spread of the ASFV in the world and the lack of commercial vaccines, the development of improved control strategies, including antiviral drugs, is urgently needed. BRD4 is an important epigenetic factor and has been commonly used for drug development for tumor treatment. Furthermore, the latest research showed that BET/BRD4 inhibition could suppress replication of virus. In this study, we first showed the inhibitory effect of agents targeting BET/BRD4 on ASFV infection with no significant host cytotoxicity. Then, we found four BET/BRD4 inhibitors that can inhibit ASFV replication, RNA transcription, and protein synthesis. Our findings support the hypothesis that BET/BRD4 can be considered as attractive host targets in antiviral drug discovery against ASFV.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Febre Suína Africana/genética , Febre Suína Africana/prevenção & controle , Vírus da Febre Suína Africana/genética , Animais , Antivirais/farmacologia , Epigênese Genética , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Acute myeloid leukemia (AML) is a heterogeneous neoplasm characterized by variations in cytogenetics and molecular abnormalities, which result in variable response to therapy. Receptor-interacting serine/threonine kinase 1 (RIP1)-mediated necroptosis has been reported to have a potential role in the treatment of AML. We obtained Skp2 and RIP1 are significantly overexpressed in AML samples using original published data, and identified that Skp2-depletion in AML cells significantly suppressed RIP1. Functional analysis showed that the inhibition of RIP1 caused by necrostatin-1 (Nec-1) inhibited the proliferation, simultaneously facilitate both the apoptosis and differentiation of AML cells. Mechanistical analysis elucidated that knockdown of Skp2 suppresses RIP1 by transcriptional regulation but not by proteasome degradation. Additionally, Skp2 regulated the function of RIP1 by decreasing K63-linked ubiquitin interaction with RIP1. Moreover, the suppression of Akt/GSK3ß was observed in Skp2 knockdown stable NB4 cells. Also, GSK3ß inactivation via small-molecule inhibitor treatment remarkably decreased RIP1 level. RIP1 regulates differentiation by interacting with RARα, increasing RA signaling targets gene C/EBPα and C/EBPß. In conclusion, our study provides a novel insight into the mechanism of tumorigenesis and the development of AML, for which the Skp2-Akt/GSK3ß-RIP1 pathway can be developed as a promising therapeutic target.