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UVB radiation induces inflammatory and oxidative stress responses, contributing to skin damage, yet the underlying mechanisms are not fully understood. N-Myc downstream-regulated gene 2 (NDRG2), an emerging stress-associated gene, remains unexplored in UVB-induced skin injury. In this study, we detected skin NDRG2 expression after UVB irradiation for the first time and further used Ndrg2 knockout mice to clarify the role of NDRG2 in UVB-induced skin injury. Three-month-old male Ndrg2+/+ and Ndrg2-/- mice (16-18g) were exposed to UVB to induce acute skin damage, and then dorsal skin samples were collected for subsequent analyses. UVB-induced skin damage was scored. Western Blot Analysis, immunofluorescence (IF) double labeling, and immunohistochemistry (IHC) were employed to assess NDRG2 expression and/or distribution. The concentrations of TNF-α, IL-6, IL-1ß, MPO, MMP8, superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) were quantitatively assessed using enzyme-linked immunosorbent assay (ELISA). Hematoxylin and eosin (HE) staining were employed to determine pathological changes. RNA sequencing and analysis were performed to estimate transcript expression levels and analyze mRNA expression. DESeq2 software was employed to identify differentially expressed genes (DEGs). DEGs were visualized using volcanic and heat maps. Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed to identify primary biological functions, metabolic pathways, or signal transduction pathways associated with DEGs. UVB-challenged Ndrg2-/- mice exhibited significantly exacerbated skin damage (erythema, edema, and erosion), neutrophil infiltration, and apoptosis compared to Ndrg2+/+ mice. Furthermore, UVB-challenged Ndrg2-/- mice displayed significantly elevated pro-inflammatory cytokines, myeloperoxidase (MPO), matrix metalloproteinase-8 (MMP8), and reduced antioxidant expression. RNA sequencing identified 1091 significantly differentially expressed genes enriched in inflammation, immune response, and oxidative stress pathways. In conclusion, the deficiency of Ndrg2 markedly exacerbated UVB-induced skin damage by promoting inflammatory responses and inhibiting antioxidant responses. This suggests that stabilizing NDRG2 expression holds promise as a therapeutic strategy for protecting against UVB-induced skin damage.
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Ischemic stroke is one main type of cerebrovascular disorders with leading cause of death and disability worldwide. Astrocytes are the only nerve cell type storing glycogen in the brain, which regulate the glucose metabolism and handle the energy supply and survive of neurons. Astrocyte ferroptosis contributes to neuron injury in brain disorders. N-myc downstream-regulated gene 2 (NDRG2) has been implicated in the progression of brain diseases, including ischemic stroke. However, whether NDRG2 could affect the glucose metabolism and ferroptosis of astrocytes during ischemic stroke remains largely unknown. Mouse astrocytes were treated with oxygen-glucose deprivation/reoxygenation (OGD/R) to establish the in vitro model. Glial fibrillary acidic protein, NDRG2, Wnt3a and ß-catenin expression levels were detected by immunofluorescence staining and western blot analyses. Glucose metabolism was investigated by glucose uptake, lactate production, nicotinamide adenine dinucleotide phosphate hydrogen/nicotinamide adenine dinucleotide phosphate (NADPH/NADP+), ATP and glycolysis enzymes (HK2, PKM2 and lactate dehydrogenase A [LDHA]) levels. Ferroptosis was assessed via reactive oxygen species (ROS), glutathione (GSH), iron and ferroptosis-related markers (GPX4 and PTGS2) contents. Glycolysis enzymes and ferroptosis-related markers levels were measured via western blot. NDRG2 expression was elevated in OGD/R-induced astrocytes. NDRG2 overexpression aggravated OGD/R-induced loss of glucose metabolism through reducing glucose uptake, lactate production, NADPH/NADP+ and ATP levels. NDRG2 upregulation exacerbated OGD/R-caused reduction of glycolysis enzymes (HK2, PKM2 and LDHA) levels. NDRG2 promoted OGD/R-induced ferroptosis of astrocytes by increasing ROS, iron and PTGS2 levels and decreasing GSH and GPX4 levels. NDRG2 overexpression enhanced OGD/R-induced decrease of Wnt/ß-catenin signaling activation by reducing Wnt3a and ß-catenin expression. NDRG2 silencing played an opposite effect. Inhibition of Wnt/ß-catenin signaling activation by IWR-1 attenuated the influences of NDRG2 knockdown on glucose metabolism, glycolysis enzymes levels and ferroptosis. These findings demonstrated that NDRG2 contributes to OGD/R-induced inhibition of glucose metabolism and promotion of ferroptosis in astrocytes through inhibiting Wnt/ß-catenin signaling activation, which might be associated with ischemic stroke progression.
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Astrocitos , Ferroptosis , Glucosa , Vía de Señalización Wnt , beta Catenina , Astrocitos/metabolismo , Animales , Glucosa/metabolismo , Ratones , beta Catenina/metabolismo , Glucólisis , Especies Reactivas de Oxígeno/metabolismo , Células Cultivadas , Oxígeno/metabolismo , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
Salvianolic acid B (Sal B) has demonstrated anticancer activity against various types of cancer. However, the underlying mechanism of Sal B-mediated anticancer effects remains incompletely understood. This study aims to investigate the impact of Sal B on the growth and metastasis of human A549 lung cells, as well as elucidate its potential mechanisms. In this study, different concentrations of Sal B were administered to A549 cells. The effects on migration and invasion abilities were assessed using MTT, wound healing, and transwell assays. Flow cytometry analysis was employed to evaluate Sal B-induced apoptosis in A549 cells. Western blotting and immunohistochemistry were conducted to measure the expression levels of cleaved caspase-3, cleaved PARP, and E-cadherin. Commercial kits were utilized for detecting intracellular reactive oxygen species (ROS) and NAD+. Additionally, a xenograft model with transplanted A549 tumors was employed to assess the anti-tumor effect of Sal B in vivo. The expression levels of NDRG2, p-PTEN, and p-AKT were determined through western blotting. Our findings demonstrate that Sal B effectively inhibits proliferation, migration, and invasion in A549 cells while inducing dose-dependent apoptosis. These apoptotic responses and inhibition of tumor cell metastasis are accompanied by alterations in intracellular ROS levels and NAD+/NADH ratio. Furthermore, our in vivo experiment reveals that Sal B significantly suppresses A549 tumor growth compared to an untreated control group while promoting increased cleavage of caspase-3 and PARP. Importantly, we observe that Sal B upregulates NDRG2 expression while downregulating p-PTEN and p-AKT expressions. Collectively, our results provide compelling evidence supporting the ability of Sal B to inhibit both growth and metastasis in A549 lung cancer cells through oxidative stress modulation as well as involvement of the NDRG2/PTEN/AKT pathway.
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Benzofuranos , Movimiento Celular , Proliferación Celular , Neoplasias Pulmonares , Estrés Oxidativo , Fosfohidrolasa PTEN , Transducción de Señal , Humanos , Fosfohidrolasa PTEN/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células A549 , Animales , Proliferación Celular/efectos de los fármacos , Benzofuranos/farmacología , Movimiento Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ratones , Apoptosis/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones Desnudos , Ratones Endogámicos BALB C , Especies Reactivas de Oxígeno/metabolismo , DepsidosRESUMEN
Diffuse large B-cell lymphoma (DLBCL) represents the most common tumor in non-Hodgkin's lymphoma. N-Myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor highly expressed in healthy tissues but downregulated in many cancers. Although cell proliferation-related metabolism rewiring has been well characterized, less is known about the mechanism of metabolic changes with DLBCL. Herein, we investigated the expressions of NDRG2, MYC and Myc-interacting zinc finger protein 1 (MIZ-1) in seven human lymphoma (mostly DLBCLs) cell lines. NDRG2 expression was inversely correlated with the expressions of MYC and MIZ-1. Further, we explored the regulatory mechanism and biological functions underlying the lymphomagenesis involving NDRG2, MYC and MIZ-1. MYC and MIZ-1 promoted DLBCL cell proliferation, while NDRG2 induced apoptosis in LY8 cells. Moreover, NDRG2 methylation was reversed by the 5-Aza-2'-deoxycytidine (5-Aza-CDR) treatment, triggering the downregulation of MYC and inhibiting DLBCL cell survival. MYC interacts with NDRG2 to regulate energy metabolism associated with mTOR. Remarkably, supporting the biological significance, the converse correlation between NDRG2 and MYC was observed in human DLBCL tumor tissues (R = -0.557). Bioinformatics analysis further validated the association among NDRG2, MYC, MIZ-1, mTOR, and related metabolism genes. Additionally, NDRG2 (P = 0.001) and MYC (P < 0.001) were identified as promising prognostic biomarkers in DLBCL patients through survival analysis. Together, our data demonstrate that the MYC/MIZ-1 complex interplays with NDRG2 to influence the proliferation and apoptosis of DLBCL cells and show the characterizations of NDRG2, MYC and MIZ-1 for metabolism features and prediction prognosis in DLBCL.
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Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso , Proteínas Proto-Oncogénicas c-myc , Proteínas Supresoras de Tumor , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Masculino , Pronóstico , Línea Celular Tumoral , Femenino , Persona de Mediana Edad , Metilación de ADN , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Anciano , Proliferación Celular , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismoRESUMEN
The protein, N-myc downstream-regulated gene 2 (NDRG2), a tumor suppressor, is significantly decreased or absent in many types of cancer. There is a significant negative correlation between the levels of NDRG2 and the development and progression of cancer tumor recurrence and tumor invasion, in different cancers. In contrast, the in vitro and in vivo overexpression of the NDRG2 protein decreases the proliferation, growth, adhesion and migration of many types of cancer cells. The in vitro overexpression of NDRG2 increases the efficacy of certain anticancer drugs in specific types of cancer cells. We hypothesize that the delivery of the mRNA of the NDRG2 protein, encapsulated by lipid nanoparticles, could represent a potential treatment of metastatic and drug-resistant cancers. This would be accomplished using a self-amplifying mRNA that encodes the NDRG2 protein and an RNA-dependent-RNA polymerase, obtained from an in vitrotranscribed (IVT) mRNA. The IVT mRNA would be encapsulated in a lipid nanoformulation. The efficacy of the nanoformulation would be determined in cultured cancer cells and if the results are positive, nude mice transplanted with either drug-resistant or metastatic drug-resistant cancer cells, would be treated with the nano- formulation and monitored for efficacy and adverse effects. If the appropriate preclinical studies indicate this formulation is efficacious and safe, it is possible it could be evaluated in clinical trials.
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OBJECTIVES: Gliobalstoma is the most common primary brain tumor in adults with an extensive genetic and transcriptional heterogeneity, still identification of the role of DNA methylation, as one of epigenetic alterations, is emerged. Authors aimed to study the clinical role of N-myc downstream-regulated gene 2 (NDRG2) -based methylation among GBM patients versus benign neurological diseases (BND), investigate its prognostic role and its relation with survival outcomes. METHODS: A total of 78 FFPE specimens were recruited as follows: GBM (n = 58) and BND (n = 20) then analyzed for NDRG2 methylation using Methyl II quantitative PCR system. The sensitivity and specificity of methylation was detected using receiver operating characteristic (ROC) curve and the relation with clinicopathological criteria for GBM and response to treatment were studied. Survival patterns; progression free survival (PFS) and overall survival (OS) were analyzed using Kaplan-Meier analyses. RESULTS: Mean methylation NDRG2 level was significantly increased in GBM patients as compared to BND and its sensitivity and specificity were 96.55% and 95%, respectively with area under curve (AUC) equals 0.973. Among the clinical characteristic factors, mean methylation level reported significant difference with ECOG and tumor site. Survival out comes revealed that NDRG2 methylation increased with worse PFS and OS at significant level (long rank test X2 = 13.3, p < .0001; and X2 = 7.1, p = .008, respectively). CONCLUSION: Current findings highlight the importance of studying DNA methylation of NDRG2 as a key factor to understand the role of epigenetic alterations in GBM.
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Neoplasias Encefálicas , Metilación de ADN , Glioblastoma , Proteínas Supresoras de Tumor , Humanos , Glioblastoma/genética , Glioblastoma/mortalidad , Glioblastoma/patología , Masculino , Proteínas Supresoras de Tumor/genética , Femenino , Persona de Mediana Edad , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Adulto , Pronóstico , Anciano , Biomarcadores de Tumor/genética , Epigénesis Genética , Estimación de Kaplan-MeierRESUMEN
N-myc downstream-regulated gene 2 (NDRG2), which is a tumour suppressor, is frequently lost in many types of tumours, including adult T-cell leukaemia/lymphoma (ATL). The downregulation of NDRG2 expression is involved in tumour progression through the aberrant phosphorylation of several important signalling molecules. We observed that the downregulation of NDRG2 induced the translocation of protein arginine methyltransferase 5 (PRMT5) from the nucleus to the cytoplasm via the increased phosphorylation of PRMT5 at Serine 335. In NDRG2low ATL, cytoplasmic PRMT5 enhanced HSP90A chaperone activity via arginine methylation, leading to tumour progression and the maintenance of oncogenic client proteins. Therefore, we examined whether the inhibition of PRMT5 activity is a drug target in NDRG2low tumours. The knockdown of PRMT5 and binding partner methylsome protein 50 (MEP50) expression significantly demonstrated the suppression of cell proliferation via the degradation of AKT and NEMO in NDRG2low ATL cells, whereas NDRG2-expressing cells did not impair the stability of client proteins. We suggest that the relationship between PRMT5/MEP50 and the downregulation of NDRG2 may exhibit a novel vulnerability and a therapeutic target. Treatment with the PRMT5-specific inhibitors CMP5 and HLCL61 was more sensitive in NDRG2low cancer cells than in NDRG2-expressing cells via the inhibition of HSP90 arginine methylation, along with the degradation of client proteins. Thus, interference with PRMT5 activity has become a feasible and effective strategy for promoting cancer vulnerability in NDRG2low ATL.
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Péptidos y Proteínas de Señalización Intracelular , Leucemia-Linfoma de Células T del Adulto , Linfoma , Neoplasias , Adulto , Humanos , Proteína-Arginina N-Metiltransferasas/metabolismo , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Arginina/metabolismo , Metilación , Proteínas Supresoras de Tumor/metabolismoRESUMEN
N-myc downstream-regulated gene 2 (NDRG2) has been recognised as a negative regulator of the progression of numerous tumours, yet its specific role in small-cell lung carcinoma (SCLC) is not fully understood. The purpose of the current study was to investigate the biological role and mechanism of NDRG2 in SCLC. Initial investigation using the Gene Expression Omnibus (GEO) dataset revealed marked downregulation of NDRG2 transcripts in SCLC. The decreased abundance of NDRG2 in SCLC was verified by examining clinical specimens. Increasing NDRG2 expression in SCLC cell lines caused significant changes in cell proliferation, cell cycle progression, colony formation, and chemosensitivity. NDRG2 overexpression decreased the levels of phosphorylated PTEN, AKT and mTOR. In PTEN-depleted SCLC cells, the upregulation of NDRG2 did not result in any noticeable impact on AKT or mTOR activation. Additionally, the reactivation of AKT reversed the antitumour effects of NDRG2 in SCLC cells. Notably, increasing NDRG2 expression retarded the growth of SCLC cell-derived xenografts in vivo. In conclusion, NDRG2 serves as an inhibitor of SCLC, and its cancer-inhibiting effects are achieved through the suppression of AKT/mTOR via the activation of PTEN. This work suggests that NDRG2 is a potential druggable target for SCLC treatment.
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Proliferación Celular , Neoplasias Pulmonares , Ratones Desnudos , Fosfohidrolasa PTEN , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Carcinoma Pulmonar de Células Pequeñas , Serina-Treonina Quinasas TOR , Proteínas Supresoras de Tumor , Humanos , Serina-Treonina Quinasas TOR/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Línea Celular Tumoral , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ratones , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Femenino , Masculino , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Tumor suppressor (p53) acts to integrate multiple stress signals into diverse antiproliferative responses. Its potential to transactivate or downregulate genes through apoptotic pathway in IDH-wildtype glioblastoma has never been explored. METHODS: A group of twenty patients diagnosed with IDH-wildtype glioblastoma, were tested for p53 expression and NDRG2/NRF2 genes activity through protein and gene profiling assays. The connotation between these elements has been explored. RESULTS: The mean patients' age was 64-years. All tumors were IDH-wildtype. p53 was expressed in 12 tumors and absent in 8 tumors. The activity of NDRG2 gene was downregulated in all cases. The activity of NRF2 gene was upregulated in 17 tumors and downregulated in 3 tumors. There was a significant statistical difference in PFS among tumors exhibiting different levels of p53 expression and NDRG2 gene activity [p-value= 0.025], in which 12 tumors with downregulated NDRG2 expression and positive p53 expression had earlier tumor recurrence. This statistical difference in PFS was insignificant when we compared p53 expression with NRF2 gene activity [p-value= 0.079]. CONCLUSIONS: During cell cycle arrest at G2 phase, p53 expression in IDH-wildtype glioblastoma in elderly individuals, coupled with the downregulation of NDRG2 gene activity, led to an aberrant increase in tumor cell proliferation and accelerated tumor recurrence. However, the influence of p53 on NRF2 gene activity was found to be insignificant.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Anciano , Persona de Mediana Edad , Glioblastoma/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Recurrencia Local de Neoplasia , Neoplasias Encefálicas/patología , Isocitrato DeshidrogenasaRESUMEN
OBJECT: This study aims to investigate the molecular mechanism of NDRG2 (N-myc downstream-regulated gene 2) in the cell senescence of lens endothelial cells. METHODS: Lens endothelial cells (SRA01/04) were irradiated with UVB at different times. Cell viability was measured by CCK-8 kit and cell cycle was detected by flow cytometry. Cell senescence was detected using SA-ß-gal staining. Western blot was utilized to detect the expressions of p53, p21 and NDRG2. TUNEL staining and flow cytometry were used to detect apoptosis and pyroptosis. RESULTS: UVB-irradiation significantly induces cell senescence and the expression of NDRG2, p53 and p21 in SRA01/04 cells was up-regulated. Down-regulation of NDRG2 inhibited UVB-induced cell senescence, significantly reversed pyroptosis and promoted cell proliferation. UVB-induced pyroptosis is closely related to caspase-1/NLRP3 axis. CONCLUSION: Our study confirmed downregulation of NDRG2 significantly inhibited UVB radiation-induced cell senescence by regulating caspase-1/NLRP3-mediated pyroptosis.
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Caspasa 1 , Senescencia Celular , Células Epiteliales , Cristalino , Proteína con Dominio Pirina 3 de la Familia NLR , Piroptosis , Rayos Ultravioleta , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Senescencia Celular/efectos de la radiación , Caspasa 1/metabolismo , Células Epiteliales/metabolismo , Cristalino/citología , Cristalino/metabolismo , Cristalino/efectos de la radiación , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Humanos , Línea Celular , Proliferación CelularRESUMEN
The aim of the present study was to evaluate the effect of ETS homologous factor (EHF) in malignant breast cancer cells. The overexpression and knockdown of the EHF gene in human and mouse breast cancer cells were performed, and the TCGA dataset and Q-omics were analyzed. We found that the tumor suppressor NDRG2 is correlated with EHF gene expression in triple-negative breast cancer cells, that EHF overexpression results in reduced cell proliferation and that apoptosis is promoted by the chemotherapeutic reagent treatment of EHF-overexpressing cells. By EHF overexpression, senescence-associated ß-galactosidase activity and p21WAF1/CIP1 expression were increased, suggesting that EHF may induce cellular senescence. In addition, the overexpression of EHF reduced the migratory ability and inhibited epithelial-mesenchymal transition (EMT). Furthermore, EHF inhibited the phosphorylation of STAT3. The overexpression of EHF also reduced the tumor size, and lung metastasis in vivo. At the tumor site, ß-galactosidase activity was increased by EHF. Finally, the Kaplan-Meier-plotter analysis showed that TNBC patients with a high expression of EHF had a longer relapse-free survival rate. Our findings demonstrated that EHF inhibits breast tumor progression by inducing senescence and regulating EMT in TNBC cells.
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BACKGROUND: NDRG2 is a tumour suppressor gene involved in tumor growth inhibition. Its effect on tumour recurrence remains controversial. The aim of this study is to explore the dual effect of IDH mutation and NDRG2 dysregulation in WHO-Grade 4 astrocytoma recurrence. METHODS: A group of 36 patients with WHO-Grade 4 astrocytoma were examined for NDRG2 expression using protein and gene expression assays. The relationship between IDH, NDRG2 protein and gene expressions, and recurrence-free interval [RFI] was explored. RESULTS: The mean patients age in this study was 45-years with 21 males and 15 females. IDH was mutant in 22 tumors. NDRG2 protein expression was low in 23 tumors, and high in 13 tumors. NDRG2 gene expression was upregulated in 4 tumors and 32 tumors showed NDRG2 gene downregulation. The consistency between two tasting methods of NDRG2 expression was 52.8%. There was a significant statistical difference in RFI among tumors with varying NDRG2 gene expression and IDH mutation [p-value= 0.021]. IDH-mutant tumours with downregulated NDRG2 expression showed late recurrence compared to IDH-wildtype glioblastoma. CONCLUSIONS: IDH-mutant WHO Grade-4 astrocytoma with downregulated NDRG2 gene are associated with late tumor recurrence. IDH mutations cause excessive accumulation of D-2-hydroxyglutarate, that may inhibit the activity of TET proteins, potentially leading to DNA hypermethylation and gene silencing.
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Damages of sensory hair cells (HCs) are mainly responsible for sensorineural hearing loss, however, its pathological mechanism is not yet fully understood due to the fact that many potential deafness genes remain unidentified. N-myc downstream-regulated gene 2 (ndrg2) is commonly regarded as a tumor suppressor and a cell stress-responsive gene extensively involved in cell proliferation, differentiation, apoptosis and invasion, while its roles in zebrafish HC morphogenesis and hearing remains unclear. Results of this study suggested that ndrg2 was highly expressed in the HCs of the otic vesicle and neuromasts via in situ hybridization and single-cell RNA sequencing. Ndrg2 loss-of-function larvae showed decreased crista HCs, shortened cilia, and reduced neuromasts and functional HCs, which could be rescued by the microinjection of ndrg2 mRNA. Moreover, ndrg2 deficiency induced attenuated startle response behaviors to sound vibration stimuli. Mechanistically, there were no detectable HC apoptosis and supporting cell changes in the ndrg2 mutants, and HCs were capable of recovering by blocking the Notch signaling pathway, suggesting that ndrg2 was implicated in HC differentiation mediated by Notch. Overall, our study demonstrates that ndrg2 plays crucial roles in HC development and auditory sensory function utilizing the zebrafish model, which provides new insights into the identification of potential deafness genes and regulation mechanism of HC development.
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Sordera , Proteínas Supresoras de Tumor , Proteínas de Pez Cebra , Pez Cebra , Animales , Proliferación Celular , Sordera/metabolismo , Células Ciliadas Auditivas/metabolismo , Audición , Neurogénesis/genética , Pez Cebra/genética , Proteínas Supresoras de Tumor/genética , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND: Dementia is a serious complication in patients with diabetes-associated cognitive dysfunction (DACD). In this study, we aim to explore the protective effect of exercise on DACD in diabetic mice, and the role of NDRG2 as a potential guarder for reversing the pathological structure of neuronal synapses. METHODS: Seven weeks of standardized exercise at moderate intensity was carried out using an animal treadmill in the vehicle + Run and STZ + Run groups. Based on quantitative transcriptome and tandem mass tag (TMT) proteome sequencing, weighted gene co-expression analysis (WGCNA) and gene set enrichment analysis (GSEA) were used to investigate the activation of complement cascades to injury neuronal synaptic plasticity. Golgi staining, Western blotting, immunofluorescence staining, and electrophysiology were used to verify the reliability of sequencing data. The role of NDRG2 was assessed by overexpressing or inhibiting the NDRG2 gene in vivo. Moreover, we estimated the cognitive function in diabetic or normal patients using DSST scores. FINDINGS: Exercise reversed the injury of neuronal synaptic plasticity and the downregulation of astrocytic NDRG2 in diabetic mice, which succeeded in attenuating DACD. The deficiency of NDRG2 aggravated the activation of complement C3 by accelerating the phosphorylation of NF-κB, ultimately leading to synaptic injury and cognitive dysfunction. Conversely, the overexpression of NDRG2 promoted astrocytic remodeling by inhibiting complement C3, thus attenuating synaptic injury and cognitive dysfunction. Meanwhile, C3aR blockade rescued dendritic spines loss and cognitive deficits in diabetic mice. Moreover, the average DSST score of diabetic patients was significantly lower than that of non-diabetic peers. Levels of complement C3 in human serum were elevated in diabetic patients compared to those in non-diabetic patients. INTERPRETATION: Our findings illustrate the effectiveness and integrative mechanism of NDRG2-induced improvement of cognition from a multi-omics perspective. Additionally, they confirm that the expression of NDRG2 is closely related to cognitive function in diabetic mice and the activation of complement cascades accelerated impairment of neuronal synaptic plasticity. NDRG2 acts as a regulator of astrocytic-neuronal interaction via NF-κB/C3/C3aR signaling to restore synaptic function in diabetic mice. FUNDING: This study was supported by the National Natural Science Foundation of China (No. 81974540, 81801899, 81971290), the Key Research and Development Program of Shaanxi (Program No. 2022ZDLSF02-09) and Fundamental Research Funds for the Central Universities (Grant No. xzy022019020).
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Disfunción Cognitiva , Diabetes Mellitus Experimental , Humanos , Ratones , Animales , FN-kappa B/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Complemento C3 , Reproducibilidad de los Resultados , Disfunción Cognitiva/genética , Disfunción Cognitiva/complicaciones , Proteínas Supresoras de TumorRESUMEN
Background: N-Myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor, highly expressed in normal tissues but downregulated in many cancers. However, it has been shown to be involved in regulating glycolytic enzymes in clear cell renal cell carcinoma and colorectal cancer, although the mechanism is still not clear, and the function of NDRG2 in liver tumor glycolysis is completely unknown. Methods: Liver tumor tissues were obtained from resected tumors and confirmed by pathological review. Immunohistochemical staining was performed to assess the protein expression of NDRG2. NDRG2-overexpressed and knockdown HepG2/SMMC-7721 cell lines were infected by lentivirus and cultured, before measurement of glucose uptake, lactate production, lactase dehydrogenase activity, and oxygen consumption rate. NDRG2 and SIRT1 proteins were analyzed by western blot. Results: Both the mRNA and protein levels of NDRG2 as a tumor suppressor were downregulated in the liver tumors and the survival rate of patients negatively correlated with the expression of NDRG2. In the NDRG2-overexpressed and knockdown cell lines, NDRG2 showed inhibition of glycolysis in liver tumor cells. Our experimental data suggested the expression of SIRT1 negatively correlated with that of NDRG2. Conclusions: Our study findings enrich the understanding of the role of NDRG2 in tumor growth and of the mechanism by which NDRG2 regulates glycolysis. SIRT1, a deacetylase that plays an important role in glycolysis regulation, may be negatively regulated by NDRG2 in liver tumors.
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In response to traumatic brain injury, a subpopulation of cortical astrocytes is activated, resulting in acquisition of stem cell properties, known as reactive astrocytes-derived progenitor cells (Rad-PCs). However, the underlying mechanisms remain largely unknown during this process. In this study, we examined the role of N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, in Rad-PCs after cortical stab injury in adult rats. Immunohistochemical analysis showed that in the cerebral cortex of normal adult rats, NDRG2 was exclusively expressed in astrocytes. After liu cortical injury, the expression of NDRG2 was significantly elevated around the wound and most cells expressing NDRG2 also expressed GFAP, a reactive astrocyte marker. Importantly, NDRG2-expressing cells were co-labeled with Nestin, a marker for neural stem cells, some of which also expressed cell proliferation marker Ki67. Overexpression of NDRG2 further increased the number of NDRG2/Nestin double-labeling cells around the lesion. In contrast, shRNA knockdown of NDRG2 decreased the number of NDRG2+/Nestin+ cells. Intracerebroventricular administration of stab-injured rats with a Notch antagonist, DAPT, led to a significant decrease in Nestin+/NDRG2+ cells around the injured boundary, but did not affect NDRG2+ cells. Moreover, overexpression or knockdown of NDRG2 led to up- and down-regulation of the expression of Notch intracellular domain NICD and Notch target gene Hes1, respectively. Taken together, these results suggest that NDRG2 may play a role in controlling the formation of Rad-PCs in the cerebral cortex of adult rats following traumatic injury, and that Notch signaling pathway plays a key role in this process.
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Renal fibrosis is the common histopathological feature of chronic kidney diseases (CKD), and there is increasing evidence that epigenetic regulation is involved in the occurrence and progression of renal fibrosis. N-myc downstream-regulated gene 2 (NDRG2) is significantly down-regulated in renal fibrosis, the mechanism of which remains unclear. Previous studies have confirmed that the inhibition of NDRG2 expression in tumor cells is related to hyper-methylation, mainly regulated by DNA methyltransferases (DNMTS). Herein, we explored the expression of NDRG2 and its epigenetic regulatory mechanism in renal fibrosis. The results showed that the expression of NDRG2 was significantly inhibited in vivo and in vitro, while the overexpression of NDRG2 effectively alleviated renal fibrosis. Meanwhile, we found that the expression of DNMT1/3A/3B was significantly increased in hypoxia-induced HK2 cells and Unilateral Ureteral Obstruction (UUO) mice accompanied by hyper-methylation of the NDGR2 promoter. Methyltransferase inhibitor (5-AZA-dC) corrected the abnormal expression of DNMT1/3A/3B, reduced the methylation level of NDRG2 promoter and restored the expression of NDRG2. The upstream events that mediate changes in NDRG2 methylation were further explored. Reactive oxygen species (ROS) are important epigenetic regulators and have been shown to play a key role in renal injury due to various causes. Accordingly, we further explored whether ROS could induce DNA-epigenetic changes of the expression of NDRG2 and then participated in the development of renal fibrosis. Our results showed that mitochondria-targeted antioxidants (Mito-TEMPO) could reverse the epigenetic inhibition of NDRG2 in a DNMT-sensitive manner, showing strong ability of DNA demethylation, exhibiting epigenetic regulation and anti-fibrosis effects similar to 5-AZA-dC. More importantly, the anti-fibrotic effects of 5-AZA-dC and Mito-TEMPO were eliminated in HK2 cells with NDRG2 knockdown. These findings highlight that targeting ROS-mediated hyper-methylation of NDRG2 promoter is a potentially effective therapeutic strategy for renal fibrosis, which will provide new insights into the treatment of CKD.
Asunto(s)
ADN de Forma A , Insuficiencia Renal Crónica , Animales , Ratones , Epigénesis Genética , Especies Reactivas de Oxígeno , Metiltransferasas/genética , Metilación de ADN , Fibrosis , Insuficiencia Renal Crónica/patología , Azacitidina/uso terapéuticoRESUMEN
The regeneration of hair cells in zebrafish is a complex process involving the precise regulation of multiple signaling pathways, but this complicated regulatory network is not fully understood. Current research has primarily focused on finding molecules and pathways that can regulate hair cell regeneration and restore hair cell functions. Here, we show the role of N-Myc downstream regulated gene 2 (ndrg2) in zebrafish hair cell regeneration. We first found that ndrg2 was dynamically expressed in neuromasts of the developing zebrafish, and this expression was increased after neomycin-induced hair cell damage. Then, ndrg2 loss-of-function larvae showed reduced numbers of regenerated hair cells but increased numbers of supporting cells after neomycin exposure. By in situ hybridization, we further observed that ndrg2 loss of function resulted in the activation of Notch signaling and downregulation of atoh1a during hair cell regeneration in vivo. Additionally, blocking Notch signaling rescued the number of regenerated hair cells in ndrg2-deficient larvae. Together, this study provides evidence for the role of ndrg2 in regulating hair cell regeneration in zebrafish neuromasts.
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Sistema de la Línea Lateral , Pez Cebra , Animales , Cabello , Células Ciliadas Auditivas/metabolismo , Sistema de la Línea Lateral/metabolismo , Neomicina/farmacología , Neomicina/metabolismo , Transducción de Señal , Pez Cebra/genéticaRESUMEN
BACKGROUND: Astrocytic N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, has been involved in the cause of ischemic stroke (IS). However, its downstream effector in IS remains unclear. This study aimed to characterize expression of NDRG2 in IS patients and rats and to investigate the underlying mechanism. METHODS: The protein expression of NDRG2 and mammalian target of the rapamycin (mTOR) and the extent of mTOR phosphorylation in plasma of IS patients were detected by ELISA. An oxygen-glucose deprivation model was established in mouse neuronal cells CATH.a, followed by cell counting kit-8, flow cytometry, TUNEL, and western blot assays to examine cell viability, apoptosis and autophagy. Finally, the effect of NDRG2-mediated phosphatidylinositol 3-kinase/protein kinase-B/mTOR (PI3K/AKT/mTOR) pathway on neuronal apoptosis and autophagy was verified in rats treated with middle cerebral artery occlusion. RESULTS: NDRG2 was highly expressed in the plasma of IS patients, while the extent of mTOR phosphorylation was reduced in IS patients. NDRG2 blocked the PI3K/Akt/mTOR signaling through dephosphorylation. Depletion of NDRG2 suppressed apoptosis and autophagy in CATH.a cells, which was reversed by a dual inhibitor of PI3K and mTOR, BEZ235. In vivo experiments confirmed that NDRG2 promoted neuronal apoptosis and autophagy by dephosphorylating and blocking the PI3K/Akt/mTOR signaling. CONCLUSION: The present study has shown that NDRG2 impairs the PI3K/Akt/mTOR pathway via dephosphorylation to promote neuronal apoptosis and autophagy in IS. These findings provide potential targets for future clinical therapies for IS.
Asunto(s)
Accidente Cerebrovascular Isquémico , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratones , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Apoptosis , Autofagia , Mamíferos/metabolismo , Proteínas del Tejido NerviosoRESUMEN
BACKGROUND: Brain lactate concentrations are enhanced in response to cerebral ischemia and promote the formation of reactive astrocytes, which are major components of the neuroinflammatory response and functional recovery, following cerebral ischemia. NDRG2 is upregulated during reactive astrocyte formation. However, its regulation and function are unclear. We studied the relationship between lactate and NDRG2 in astrocytes under conditions of ischemia or oxygen-glucose deprivation (OGD). METHODS: We examined astrocytic NDRG2 expression after middle cerebral artery occlusion (MCAO) using western blot and immunofluorescence staining. Under hypoxia conditions, we added exogenous L-lactate sodium (lactate) to cultured primary astrocytes to explore the effects of lactate on the ubiquitination modification of NDRG2. We profiled the transcriptomic features of NDRG2 silencing in astrocytes after 8 h of OGD conditions as well as exogenous lactate treatment by performing RNA-seq. Finally, we evaluated the molecular mechanisms of NDRG2 in regulating TNFα under OGD conditions using western blot and immunohistochemistry. RESULTS: Reactive astrocytes strongly expressed NDRG2 in a rat model of MCAO. We also showed that lactate stabilizes astrocytic NDRG2 by inhibiting its ubiquitination. NDRG2 inhibition in astrocytes increased inflammation and upregulated immune-associated genes and signaling pathways. NDRG2 knockdown induced TNFα expression and secretion via c-Jun phosphorylation. CONCLUSIONS: We revealed that under OGD conditions, lactate plays an important anti-inflammatory role and inhibits TNFα expression by stabilizing NDRG2, which is beneficial for neurological functional recovery. NDRG2 may be a new therapeutic target for cerebral ischemia.