RESUMEN
Overall health relies on features of skeletal muscle that generally decline with age, partly due to mechanisms associated with mitochondrial redox imbalance and bioenergetic dysfunction. Previously, aged mice genetically devoid of the mitochondrial NAD(P)+ transhydrogenase (NNT, encoded by the nicotinamide nucleotide transhydrogenase gene), an enzyme involved in mitochondrial NADPH supply, were shown to exhibit deficits in locomotor behavior. Here, by using young, middle-aged, and older NNT-deficient (Nnt-/-) mice and age-matched controls (Nnt+/+), we aimed to investigate how muscle bioenergetic function and motor performance are affected by NNT expression and aging. Mice were subjected to the wire-hang test to assess locomotor performance, while mitochondrial bioenergetics was evaluated in fiber bundles from the soleus, vastus lateralis and plantaris muscles. An age-related decrease in the average wire-hang score was observed in middle-aged and older Nnt-/- mice compared to age-matched controls. Although respiratory rates in the soleus, vastus lateralis and plantaris muscles did not significantly differ between the genotypes in young mice, the rates of oxygen consumption did decrease in the soleus and vastus lateralis muscles of middle-aged and older Nnt-/- mice. Notably, the soleus, which exhibited the highest NNT expression level, was the muscle most affected by aging, and NNT loss. Additionally, histology of the soleus fibers revealed increased numbers of centralized nuclei in older Nnt-/- mice, indicating abnormal morphology. In summary, our findings suggest that NNT expression deficiency causes locomotor impairments and muscle dysfunction during aging in mice.
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Envejecimiento , Metabolismo Energético , Mitocondrias Musculares , Músculo Esquelético , Animales , Envejecimiento/metabolismo , Envejecimiento/fisiología , Ratones , Músculo Esquelético/metabolismo , Mitocondrias Musculares/metabolismo , Masculino , NADP Transhidrogenasa AB-Específica/metabolismo , NADP Transhidrogenasa AB-Específica/genética , Consumo de Oxígeno/fisiología , Ratones Noqueados , Ratones Endogámicos C57BL , Proteínas MitocondrialesRESUMEN
BACKGROUND: Pathogenic variants in the nicotinamide nucleotide transhydrogenase gene (NNT) are a rare cause of primary adrenal insufficiency (PAI), as well as functional impairment of the gonads. OBJECTIVE: Despite the description of different homozygous and compound heterozygous NNT variants in PAI patients, the extent to which the function and expression of the mature protein are compromised remains to be clarified. DESIGN: The activity and expression of mitochondrial NAD(P)+ transhydrogenase (NNT) were analyzed in blood samples obtained from patients diagnosed with PAI due to genetically confirmed variants of the NNT gene (n = 5), heterozygous carriers as their parents (n = 8), and healthy controls (n = 26). METHODS: NNT activity was assessed by a reverse reaction assay standardized for digitonin-permeabilized peripheral blood mononuclear cells (PBMCs). The enzymatic assay was validated in PBMC samples from a mouse model of NNT absence. Additionally, the PBMC samples were evaluated for NNT expression by western blotting and reverse transcription quantitative polymerase chain reaction and for mitochondrial oxygen consumption. RESULTS: NNT activity was undetectable (<4% of that of healthy controls) in PBMC samples from patients, independent of the pathogenic genetic variant. In patients' parents, NNT activity was approximately half that of the healthy controls. Mature NNT protein expression was lower in patients than in the control groups, while mRNA levels varied widely among genotypes. Moreover, pathogenic NNT variants did not impair mitochondrial bioenergetic function in PBMCs. CONCLUSIONS: The manifestation of PAI in NNT-mutated patients is associated with a complete lack of NNT activity. Evaluation of NNT activity can be useful to characterize disease-causing NNT variants.
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Enfermedad de Addison , NADP Transhidrogenasas , Animales , Humanos , Ratones , Leucocitos Mononucleares/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , NAD , NADP Transhidrogenasa AB-Específica/genética , NADP Transhidrogenasa AB-Específica/metabolismo , NADP Transhidrogenasas/genética , NADP Transhidrogenasas/metabolismoRESUMEN
Nicotinamide nucleotide transhydrogenase (NNT) deficiency causes primary adrenal insufficiency (PAI) and possibly some extra-adrenal manifestations. A limited number of these patients were previously described. We present the clinical and genetic characteristics of three family members with a biallelic novel pathogenic variant in the NNT gene. The patients were followed until the ages of 21.6, 20.2, and 4.2 years. PAI was diagnosed in the eldest two brothers after an Addisonian crisis and the third was diagnosed at the age of 4.5 months in the asymptomatic stage due to the genetic screening of family members. Whole exome sequencing with a targeted interpretation of variants in genes related to PAI was performed in all the patients. The urinary steroid metabolome was determined by gas chromatography-mass spectrometry in the asymptomatic patient. The three patients, who were homozygous for c.1575dup in the NNT gene, developed isolated glucocorticoid deficiency. The urinary steroid metabolome showed normal excretion of cortisol metabolites. The adolescent patients had slow pubertal progression with low-normal testicular volume, while testicular endocrine function was normal. Bone mineral density was in the range for osteopenia in both grown-up siblings. Echocardiography revealed no structural or functional heart abnormalities. This article is among the first with a comprehensive and chronologically-detailed description of patients with NNT deficiency.
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Enfermedad de Addison , NADP Transhidrogenasa AB-Específica/genética , NADP Transhidrogenasas , Adolescente , Preescolar , Estudios de Seguimiento , Humanos , Lactante , Masculino , Proteínas Mitocondriales/genética , NADP Transhidrogenasas/genética , Hermanos , Esteroides , Adulto JovenRESUMEN
Significance: Proton-translocating NAD(P)+ transhydrogenase, also known as nicotinamide nucleotide transhydrogenase (NNT), catalyzes a reversible reaction coupling the protonmotive force across the inner mitochondrial membrane and hydride (H-, a proton plus two electrons) transfer between the mitochondrial pools of NAD(H) and NADP(H). The forward NNT reaction is a source of NADPH in the mitochondrial matrix, fueling antioxidant and biosynthetic pathways with reductive potential. Despite the greater emphasis given to the net forward reaction, the reverse NNT reaction that oxidizes NADPH also occurs in physiological and pathological conditions. Recent Advances: NNT (dys)function has been linked to various metabolic pathways and disease phenotypes. Most of these findings have been based on spontaneous loss-of-function Nnt mutations found in the C57BL/6J mouse strain (NntC57BL/6J mutation) and disease-causing Nnt mutations in humans. The present review focuses on recent advances based on the mouse NntC57BL/6J mutation. Critical Issues: Most studies associating NNT function with disease phenotypes have been based on comparisons between different strains of inbred mice (with or without the NntC57BL/6J mutation), which creates uncertainties over the actual contribution of NNT in the context of other potential genetic modifiers. Future Directions: Future research might contribute to understanding the role of NNT in pathological conditions and elucidate how NNT regulates physiological signaling through its forward and reverse reactions. The importance of NNT in redox balance and tumor cell proliferation makes it a potential target of new therapeutic strategies for oxidative-stress-mediated diseases and cancer. Antioxid. Redox Signal. 36, 864-884.
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NADP Transhidrogenasa AB-Específica , NADP Transhidrogenasas , Animales , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/metabolismo , NAD , NADP/metabolismo , NADP Transhidrogenasa AB-Específica/genética , NADP Transhidrogenasa AB-Específica/metabolismo , NADP Transhidrogenasas/genética , NADP Transhidrogenasas/metabolismo , ProtonesRESUMEN
BACKGROUND: LncRNA NNT-AS1 (NNT-AS1) has been extensively studied as the causative agent in propagation and progression of lung and bladder cancers, and cholangiocarcinoma. However, its significance in proliferation and inflammation of diabetic nephropathy is enigmatic. This study focuses on the molecular mechanisms followed by NNT-AS1 to establish diabetic nephropathy (DN) and its potential miRNA target. METHODS: Bioinformatics analysis to identify potential miRNA target of NNT-AS1 and smad4 transcription factor was conducted using LncBase and TargetScan, and was subsequently confirmed by luciferase reporter assay. Relative quantitative expression of NNT-AS1 in human glomerular mesangial cells (HGMCs) was detected through quantitative real-time PCR and WB analysis. Cell proliferation was detected through CCK-8 assay, whereas, ELISA was conducted to evaluate the expression of inflammatory cytokines. Following this, relative expression of miR-214-5p and smad4 were confirmed through qRT-PCR and western blot analysis. RESULTS: Results from the experiments manifested up-regulated levels of NNT-AS1 and smad4 in the blood samples of DN patients as well as in HGMCs, whereas, downregulated levels of miR-214-5p were measured in the HGMCs suggesting the negative correlation between NNT-AS1 and miR-214-5p. Potential binding sites of NNT-AS1 showed miR-214-5p as its direct target and NNT-AS1 as potential absorber for this microRNA, in turn increasing the expression of transcription factor smad4. CONCLUSION: The data suggests that NNT-AS1 can be positively used as a potential biomarker and indicator of DN and causes extracellular matrix (ECM) accumulation and inflammation of human mesangial cells.
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Proliferación Celular , Nefropatías Diabéticas/fisiopatología , Matriz Extracelular/metabolismo , Inflamación/fisiopatología , Células Mesangiales/citología , NADP Transhidrogenasa AB-Específica/fisiología , ARN Largo no Codificante/fisiología , Glucemia/metabolismo , Nefropatías Diabéticas/sangre , Regulación hacia Abajo , Humanos , Células Mesangiales/metabolismo , MicroARNs/sangre , MicroARNs/genética , Proteínas Mitocondriales/sangre , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/fisiología , NADP Transhidrogenasa AB-Específica/sangre , NADP Transhidrogenasa AB-Específica/genética , ARN Largo no Codificante/sangre , ARN Largo no Codificante/genética , Proteína Smad4/sangre , Proteína Smad4/genética , Regulación hacia ArribaRESUMEN
Nicotinamide nucleotide transhydrogenase (NNT) is a proton pump in the inner mitochondrial membrane that generates reducing equivalents in the form of NAPDH, which can be used for anabolic pathways or to remove reactive oxygen species (ROS). A number of studies have linked NNT dysfunction to cardiomyopathies and increased risk of atherosclerosis; however, biallelic mutations in humans commonly cause a phenotype of adrenal insufficiency, with rare occurrences of cardiac dysfunction and testicular tumours. Here, we compare the transcriptomes of the hearts, adrenals and testes from three mouse models: the C57BL/6N, which expresses NNT; the C57BL/6J, which lacks NNT; and a third mouse, expressing the wild-type NNT sequence on the C57BL/6J background. We saw enrichment of oxidative phosphorylation genes in the C57BL/B6J in the heart and adrenal, possibly indicative of an evolved response in this substrain to loss of Nnt. However, differential gene expression was mainly driven by mouse background with some changes seen in all three tissues, perhaps reflecting underlying genetic differences between the C57BL/B6J and -6N substrains.
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Aterosclerosis/genética , Cardiomiopatías/genética , Miocardio/metabolismo , NADP Transhidrogenasa AB-Específica/genética , Fosforilación Oxidativa , Glándulas Suprarrenales/metabolismo , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Testículo/metabolismoRESUMEN
Endothelial dysfunction is a critical, initiating step in the development of hypertension (HTN) and mitochondrial reactive oxygen species (ROS) are important contributors to endothelial dysfunction. Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in the nicotinamide nucleotide transhydrogenase (Nnt) gene that are associated with endothelial dysfunction and increased risk for HTN. NNT is emerging as an important enzyme that regulates mitochondrial NADPH levels and mitochondrial redox balance by supporting the thiol dependent peroxidase systems in the mitochondria. We have previously shown that the absence of NNT in C57Bl/6J animals promotes a more severe hypertensive phenotype through reductions in â¢NO and endothelial dependent vessel dilation. However, the impact of NNT on human endothelial cell function remains unclear. We utilized NNT directed shRNA in human aortic endothelial cells to test the hypothesis that NNT critically regulates mitochondrial redox balance and endothelial function in response to angiotensin II (Ang II). We demonstrate that NNT expression and activity are elevated in response to the mitochondrial dysfunction and oxidative stress associated with Ang II treatment. Knockdown of NNT led to a significant elevation of mitochondrial ROS production and impaired glutathione peroxidase and glutathione reductase activities associated with a reduction in the NADPH/NADP+ ratio. Loss of NNT also promoted mitochondrial dysfunction, disruption of the mitochondrial membrane potential, and impaired ATP production in response to Ang II. Finally, we observed that, while the loss of NNT augmented eNOS phosphorylation at Ser1177, neither eNOS activity nor nitric oxide production were similarly increased. The results from these studies clearly demonstrate that NNT is critical for the maintenance of mitochondrial redox balance and mitochondrial function. Loss of NNT and disruption of redox balance leads to oxidative stress that compromises eNOS activity that could have a profound effect on the endothelium dependent regulation of vascular tone.
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NADP Transhidrogenasas , Angiotensina II/metabolismo , Animales , Células Endoteliales/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , NADP Transhidrogenasa AB-Específica/genética , NADP Transhidrogenasas/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
NAD(P)+ transhydrogenase (NNT) is located in the inner mitochondrial membrane and catalyzes a reversible hydride transfer between NAD(H) and NADP(H) that is coupled to proton translocation between the intermembrane space and mitochondrial matrix. NNT activity has an essential role in maintaining the NADPH supply for antioxidant defense and biosynthetic pathways. In the present report, we evaluated the effects of chemical compounds used as inhibitors of NNT over the last five decades, namely, 4-chloro-7-nitrobenzofurazan (NBD-Cl), N,N'-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-l-carnitine, and rhein, on NNT activity and mitochondrial respiratory function. Concentrations of these compounds that partially inhibited the forward and reverse NNT reactions in detergent-solubilized mouse liver mitochondria significantly impaired mitochondrial respiratory function, as estimated by ADP-stimulated and nonphosphorylating respiration. Among the tested compounds, NBD-Cl showed the best relationship between NNT inhibition and low impact on respiratory function. Despite this, NBD-Cl concentrations that partially inhibited NNT activity impaired mitochondrial respiratory function and significantly decreased the viability of cultured Nnt-/- mouse astrocytes. We conclude that even though the tested compounds indeed presented inhibitory effects on NNT activity, at effective concentrations, they cause important undesirable effects on mitochondrial respiratory function and cell viability.
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Inhibidores Enzimáticos/farmacología , Mitocondrias Hepáticas/enzimología , NADP Transhidrogenasa AB-Específica/antagonistas & inhibidores , NADP Transhidrogenasa AB-Específica/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Animales , Inhibidores Enzimáticos/química , Femenino , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/genética , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , NADP Transhidrogenasa AB-Específica/genética , Consumo de Oxígeno/genéticaRESUMEN
Breast cancer is one of the most common cancers in women. This study focuses on the effects of Long non-coding RNAs (lncRNAs) NNT-AS1 on breast cancer cell growth and metastasis. Fifty-six pairs of breast cancer (BC) tissues and matched paracarcinoma tissues were obtained. The BC cell lines and normal human breast cell line were employed. NNT-AS1 in BC cells was knocked down by shRNA. Cell counting kit-8 assay (CCK-8), colony formation assay, cell cycle analysis, cell apoptosis analysis, cound healing assay, Transwell assay, cioinformatics analysis, Western blot analysis and Xenograft model were used. Quantitative real-time polymerase chain reaction (qRT-PCR) assay indicated that expression of NNT-AS1 was obviously upregulated in breast cancer tissues compared with adjacent tissues (n=56). Knockdown of NNT-AS1 could attenuate breast cancer cell viability, proliferation, invasion and migration, as well as promote cell apoptosis and induce cell cycle arrest at G0/G1 phase. ZFP36 was directly combined with NNT-AS1, and silencing of ZFP36 could rescue tumor suppression role by downregulating NNT-AS1 on cell proliferation and metastasis. Knockdown of NNT-AS1 could suppress cell growth and metastasis via interacting with ZFP36 in vivo. This study demonstrated that knockdown of NNT-AS1 had tumor-suppressive effect on breast cancer progression and metastasis via interacting with ZFP36 in vitro and in vivo, which provides a new insight into the treatment and prognosis evaluation of breast cancer.
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Neoplasias de la Mama , NADP Transhidrogenasa AB-Específica/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , MicroARNs , Proteínas Mitocondriales/genética , ARN Largo no Codificante/genética , TristetraprolinaRESUMEN
The C57BL/6J (B6J) mouse strain has been widely used as a control strain for the study of metabolic diseases and diet induced obesity (DIO). B6J mice carry a spontaneous deletion mutation in the nicotinamide nucleotide transhydrogenase (Nnt) gene eliminating exons 7-11, resulting in expression of a truncated form of Nnt, an enzyme that pumps protons across the inner mitochondrial membrane. It has been proposed that this mutation in B6J mice is associated with epigonadal fat mass and altered sensitivity to diet induced obesity. To define the role of Nnt in the development of diet induced obesity, we generated first backcross (BC1) hybrids of wild type Nnt C57BL/6NTac and mutated Nnt C57BL/6JRj [(C57BL/6NTac×C57BL/6JRj)F1×C57BL/6NTac]. Body weight gain and specific fat-pad depot mass were measured in BC1 hybrids under high fat diet conditions. Both sexes of BC1 hybrids indicate that mice with Nnt wild type allele are highly sensitive to DIO and exhibit higher relative fat mass. In summary, our data indicate that the Nnt mutation in mice is associated with sensitivity to DIO and fat mass.
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Dieta Alta en Grasa , Mutación , NADP Transhidrogenasa AB-Específica/metabolismo , Obesidad/patología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , NADP Transhidrogenasa AB-Específica/genética , Obesidad/etiología , Obesidad/metabolismo , Fenotipo , Aumento de PesoRESUMEN
The C57BL/6J and C57BL/6N mice have well-documented phenotypic and genotypic differences, including the infamous nicotinamide nucleotide transhydrogenase (Nnt) null mutation in the C57BL/6J substrain, which has been linked to cardiovascular traits in mice and cardiomyopathy in humans. To assess whether Nnt loss alone causes a cardiovascular phenotype, we investigated the C57BL/6N, C57BL/6J mice and a C57BL/6J-BAC transgenic rescuing NNT expression, at 3, 12, and 18 mo. We identified a modest dilated cardiomyopathy in the C57BL/6N mice, absent in the two B6J substrains. Immunofluorescent staining of cardiomyocytes revealed eccentric hypertrophy in these mice, with defects in sarcomere organisation. RNAseq analysis identified differential expression of a number of cardiac remodelling genes commonly associated with cardiac disease segregating with the phenotype. Variant calling from RNAseq data identified a myosin light chain kinase 3 (Mylk3) mutation in C57BL/6N mice, which abolishes MYLK3 protein expression. These results indicate the C57BL/6J Nnt-null mice do not develop cardiomyopathy; however, we identified a null mutation in Mylk3 as a credible cause of the cardiomyopathy phenotype in the C57BL/6N.
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Cardiomiopatías/genética , Quinasa de Cadena Ligera de Miosina/genética , NADP Transhidrogenasa AB-Específica/genética , Animales , Cardiomiopatías/metabolismo , Modelos Animales de Enfermedad , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL/genética , Ratones Transgénicos/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , NADP Transhidrogenasa AB-Específica/metabolismo , NADP Transhidrogenasas/genética , NADP Transhidrogenasas/metabolismo , FenotipoRESUMEN
BACKGROUND: Cholangiocarcinoma (CCA) is a serious malignant tumor. Long non-coding RNA NNT-AS1 (NNT-AS1) takes crucial roles in several tumors. So, we planned to research the roles and underlying mechanism of NNT-AS1 in CCA. RESULTS: NNT-AS1 overexpression was appeared in CCA tissues and cell lines. Proliferation was promoted by NNT-AS1 overexpression in CCLP1 and TFK1 cells. Besides, NNT-AS1 overexpression reduced E-cadherin level and raised levels of N-cadherin, vimentin, Snail and Slug. However, the opposite trend was occurred by NNT-AS1 knockdown. Further, NNT-AS1 overexpression promoted phosphatidylinositol 3 kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK)1/2 pathways. MiR-203 was sponged by NNT-AS1 and miR-203 mimic reversed the above promoting effects of NNT-AS1. Additionally, insulin-like growth factor type 1 receptor (IGF1R) and zinc finger E-box binding homeobox 1 (ZEB1) were two potential targets of miR-203. CONCLUSION: NNT-AS1 promoted proliferation, EMT and PI3K/AKT and ERK1/2 pathways in CCLP1 and TFK1 cells through down-regulating miR-203. METHODS: CCLP1 and TFK1 cells were co-transfected with pcDNA-NNT-AS1 and miR-203 mimic. Bromodeoxyuridine (BrdU), flow cytometry, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot were employed to detect roles and mechanism of NNT-AS1. Interaction between NNT-AS1 and miR-203 or miR-203 and target genes was examined through luciferase activity experiment.
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Neoplasias de los Conductos Biliares/genética , Proliferación Celular/genética , Colangiocarcinoma/genética , Transición Epitelial-Mesenquimal/genética , MicroARNs/genética , NADP Transhidrogenasa AB-Específica/genética , ARN sin Sentido/genética , ARN Largo no Codificante/genética , Antígenos CD/metabolismo , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/patología , Cadherinas/metabolismo , Línea Celular Tumoral , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patología , Regulación hacia Abajo , Técnicas de Sustitución del Gen , Técnicas de Silenciamiento del Gen , Humanos , Sistema de Señalización de MAP Quinasas/genética , Proteínas Mitocondriales/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia Snail/metabolismo , Vimentina/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
BACKGROUND: Lung cancer is the leading cause of cancer-related mortality worldwide. Studies have demonstrated that long noncoding RNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) functioned as an oncogene in most malignancies, including non-small cell lung cancer (NSCLC). This study aimed to investigate the underlying mechanisms of NNT-AS1 in NSCLC progression. METHODS: The levels of NNT-AS1, miR-22-3p and Yes-associated protein (YAP1) were detected by qRT-PCR in NSCLC tissues and cells. Kaplan-Meier analysis was conducted to analyze the correlation between NNT-AS1 expression and overall survival of NSCLC patients. Cell proliferation was evaluated by MTT assay. Cell migration and invasion were assessed using transwell assay. The protein levels of YAP1 and EMT-related proteins were detected by western blot. The molecular mechanism was predicted by starBase2.0 and validated by dual-luciferase reporter assay or RNA pull-down assay. Xenograft analysis was carried out to analyze tumor growth in vivo. RESULTS: We found that the levels of NNT-AS1 and YAP1 were enhanced, while miR-22-3p expression was decreased in NSCLC tissues and cells. High NNT-AS1 expression was correlated with poor prognosis. NNT-AS1 knockdown impeded proliferation, migration, invasion and EMT of NSCLC cells. NNT-AS1 targeted miR-22-3p, and YAP1 was a target of miR-22-3p in NSCLC cells. Furthermore, NNT-AS1 facilitated the progression of NSCLC by regulating miR-22-3p/YAP1 axis. NNT-AS1 knockdown repressed tumor growth in vivo. CONCLUSION: NNT-AS1 facilitated proliferation, migration, invasion and EMT of NSCLC cells by sponging miR-22-3p and regulating YAP1 expression, which might provide a potential biomarker and therapeutic target for NSCLC.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , MicroARNs/genética , ARN Largo no Codificante/genética , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular , Proliferación Celular , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Mitocondriales/genética , NADP Transhidrogenasa AB-Específica/genética , Pronóstico , ARN sin Sentido/genética , Factores de Transcripción/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Señalizadoras YAPRESUMEN
OBJECTIVE: Lung cancer is the main burden on human health, with high mortality and poor prognosis. The involvement of long non-coding RNAs (lncRNAs) in the development of cancer has attracted wide attention. This study aimed to investigate the role and novel mechanisms of lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in the progression of lung cancer. MATERIALS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to detect the expression of NNT-AS1, microRNA-3666 (miR-3666), and E2F transcription factor 2 (E2F2). 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to analyze cell proliferation. Flow cytometry was carried out to investigate cell apoptosis. Transwell assay was conducted to observe cell invasion. The interaction between miR-3666 and NNT-AS1 or E2F2 was predicted by bioinformatics tool starBase v2.0 and verified by Dual-Luciferase reporter assay. The protein level of E2F2 was quantified by Western blot. RESULTS: NNT-AS1 and E2F2 were upregulated, but miR-3666 was downregulated in lung cancer tissues and cells. NNT-AS1 knockdown attenuated proliferation and invasion but enhanced apoptosis of lung cancer cells, while miR-3666 inhibition reversed these effects. It was confirmed that miR-3666 was a target of NNT-AS1 and it directly interacted with E2F2. The inhibitory proliferation and invasion, and acceleratory apoptosis of lung cancer cells, caused by miR-3666 enrichment, were overturned by E2F2 overexpression. Furthermore, E2F2 was regulated by NNT-AS1 through miR-3666. CONCLUSIONS: NNT-AS1 participated in the progression of lung cancer through NNT-AS1/miR-3666/E2F2 regulatory axis at least in part. Our study supplied a promising strategy for the treatment of lung cancer.
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Factor de Transcripción E2F2/metabolismo , Neoplasias Pulmonares/metabolismo , MicroARNs/metabolismo , NADP Transhidrogenasa AB-Específica/metabolismo , ARN Largo no Codificante/metabolismo , Apoptosis , Proliferación Celular , Células Cultivadas , Factor de Transcripción E2F2/genética , Humanos , Neoplasias Pulmonares/patología , MicroARNs/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , NADP Transhidrogenasa AB-Específica/genética , ARN Largo no Codificante/genéticaRESUMEN
OBJECTIVE: The aim of the present study was to examine the effect of glucose deprivation on the expression of genes encoded glucocorticoid receptor (NR3C1) and some related proteins (NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme 1) for evaluation of their possible significance in the control of glioma growth through endoplasmic reticulum stress signaling mediated by IRE1 and glucose deprivation. METHODS: The expression of NR3C1, NR3C2, AHR, NRIP1, NNT, ARHGAP35, SGK1, and SGK3 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control cells) and cells without ERN1 signaling enzyme function (transfected by dnERN1) under glucose deprivation was studied by real time quantitative polymerase chain reaction. RESULTS: It was shown that the expression level of NR3C2, AHR, SGK1, SGK3, and NNT genes was up-regulated in control U87 glioma cells under glucose deprivation condition in comparison with the control cells growing with glucose. At the same time, the expression of NRIP1 gene is down-regulated in these glioma cells under glucose deprivation, but NR3C1 and ARHGAP35 genes was resistant to this experimental condition. We also showed that inhibition of ERN1 signaling enzyme function significantly modified the response of most studied gene expressions to glucose deprivation condition. Thus, effect of glucose deprivation on the expression level of NR3C2, AHR, and SGK1 genes was significantly stronger in ERN1 knockdown U87 glioma cells since the expression of NNT gene was resistant to glucose deprivation condition. Moreover, the inhibition of ERN1 enzymatic activities in U87 glioma cells led to up-regulation of ARHGAP35 gene expression and significant down-regulation of the expression of SGK3 gene in response to glucose deprivation condition. CONCLUSIONS: Results of this study demonstrated that glucose deprivation did not change the expression level of NR3C1 gene but it significantly affected the expression of NR3C2, AHR, NRIP, SGK1, SGK3, and NNT genes in vector-transfected U87 glioma cells in gene specific manner and possibly contributed to the control of glioma growth since the expression of most studied genes in glucose deprivation condition was significantly dependent on the functional activity of IRE1 signaling enzyme.
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Neoplasias Encefálicas/genética , Endorribonucleasas/genética , Glioma/genética , Glucosa/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Receptores de Glucocorticoides/genética , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Endorribonucleasas/deficiencia , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glioma/patología , Glucosa/farmacología , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Mitocondriales/genética , NADP Transhidrogenasa AB-Específica/genética , Proteína de Interacción con Receptores Nucleares 1/genética , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Background: Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial redox-induced proton pump that links NADPH synthesis to the mitochondrial metabolic pathway. It also participates in the regulation of immune responses. A long non-coding RNA namely NNT-antisense 1 (NNT-AS1) has been shown to be transcribed from the same locus and exert anti-proliferative effects in some tissues. Methods: In the current study, we evaluated expression of NNT and NNT-AS1 in peripheral blood of 50 relapsing-remitting multiple sclerosis patients compared with healthy subjects. The difference in NNT expression was significant in only in male subjects aged over 50 when compared with the corresponding control subgroup. Results: For NNT-AS1, based on the results of Quantile regression and adjustment of the effects of age and sex as well as the interaction between sex and disease status, no significant difference was found between cases and controls. Moreover, NNT and NNT-AS1 expressions were correlated with age in controls and in female subjects respectively. Conclusion: Finally, we assessed correlations between expressions of these genes and detected significant pairwise correlations between transcript levels of NNT and NNT-AS1 genes in both cases and controls. The current study highlights a gender-specific role for NNT in the pathogenesis of MS.
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Esclerosis Múltiple Recurrente-Remitente/sangre , NADP Transhidrogenasa AB-Específica/sangre , Adulto , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/sangre , Proteínas Mitocondriales/genética , Esclerosis Múltiple Recurrente-Remitente/enzimología , Esclerosis Múltiple Recurrente-Remitente/genética , NADP Transhidrogenasa AB-Específica/genética , ARN Largo no Codificante/sangre , ARN Largo no Codificante/genética , Caracteres SexualesRESUMEN
AIM: Nicotinamide Nucleotide Transhydrogenase (NNT) gene encodes a protein, which is an important antioxidative enzyme that converts NADH to NADPH. This enzyme provides a significant proportion of the entire NADPH resource in the mitochondria. Previous reports have shown possible contribution of this gene in the carcinogenesis process. METHODS: In the current research, we evaluated expression levels of NNT gene and a naturally occurring antisense RNA (NNT-AS1) in gastric cancer specimens compared to their corresponding adjacent noncancerous tissues (ANCTs). RESULTS: Both NNT1 and NNT-AS1 genes were significantly downregulated in tumor tissues compared to ANCTs (expression ratio = 0.369, p = .045 and expression ratio = 0.368, p = .043, respectively). Transcript levels of NNT1 and NNT-AS1 were associated with the location of the primary tumor (p = .003 and .002, respectively). Moreover, expressions of both genes were significantly elevated in tumors with lymphatic/vascular invasion compared to tumors without lymphatic/vascular invasion (p = .001 and p = .005). No other remarkable associations were noticed between transcript levels of genes in tumor tissues and patients' information. Based on the area under curve (AUC) values in the receiver operating characteristic (ROC) curves, the diagnostic power of NNT1 and NNT-AS1 were estimated to be 0.62 and 0.63, respectively. CONCLUSIONS: Although we demonstrated dysregulation of NNT1 and NNT-AS1 in gastric tumor specimens in association with clinical data of patients, these two genes are not supposed to be appropriate biomarkers for gastric cancer.
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Biomarcadores de Tumor/metabolismo , NADP Transhidrogenasa AB-Específica/metabolismo , ARN sin Sentido/metabolismo , Neoplasias Gástricas/patología , Adolescente , Adulto , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Regulación hacia Abajo , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , NADP Transhidrogenasa AB-Específica/genética , Pronóstico , ARN sin Sentido/genética , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/genética , Adulto JovenRESUMEN
Increasing studies showed that long noncoding RNAs (lncRNAs) had crucial regulatory roles in various tumors, including gastric cancer (GC). Recent studies demonstrated that lncRNA nicotinamide nucleotide transhydrogenase-antisense RNA1 (NNT-AS1) played an important role in several tumors. However, the role and expression of NNT-AS1 in GC progression remain unknown. In our study, we indicated that NNT-AS1 expression was upregulated in GC samples compared with the nontumor tissues. We also showed that NNT-AS1 expression was upregulated in the GC cell lines. Ectopic expression of NNT-AS1 promoted GC cell line HGC-27 cell proliferation, cell cycle progression, and invasion. In addition, we showed that NNT-AS1 acted as a sponge competing endogenous RNA for microRNA-363 (miR-363), which was downregulated in the GC samples and cell lines. miR-363 expression was negatively related with NNT-AS1 expression in GC samples. Upregulated expression of miR-363 suppressed GC cell growth, cycle, and invasion. Furthermore, we reported that elevated expression of NNT-AS1 promoted GC cell proliferation, cycle, and invasion partly by suppressing miR-363 expression. These results indicated that lncRNA NNT-AS1 acted as an oncogene in the development of GC partly by inhibiting miR-363 expression.
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Biomarcadores de Tumor/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , NADP Transhidrogenasa AB-Específica/antagonistas & inhibidores , ARN Largo no Codificante/genética , Neoplasias Gástricas/patología , Apoptosis , Ciclo Celular , Humanos , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , NADP Transhidrogenasa AB-Específica/genética , Invasividad Neoplásica , Pronóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To investigate the role and mechanism of action of nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in osteosarcoma (OS). METHODS: Bioinformatic analysis suggested miR-320a as potential target of NNT-AS1. Influence of NNT-AS1 overexpression or knockdown on OS cell proliferation, colony-formation, apoptosis, migration and invasion capacity was first investigated. Expression levels of NNT-AS1, miR-320a, beta-catenin, RUNX2, IGF-1R, c-Myc, Cyclin D1 and MMP13 were also evaluated by RT-qPCR and western blotting accordingly. Xenograft models using U2OS and OS-732 cells with different NNT-AS1 gene modifications were constructed for tumor formation assay as well as evaluation of miR-320a, beta-catenin and RUNX2 expression in primary lesion. NNT-AS1-overexpressing U2OS cells and NNT-AS1-knockdown OS-732 cells were subject to miR-320a mimic and inhibitor transfection, respectively, to investigate the miR-320a dependency of the osteosarcoma-promoting role of NNT-AS1. RESULTS: NNT-AS1 overexpression significantly increased proliferation, survival and mobility of U2OS cells in vitro as well as its tumor formation ability in vivo, while NNT-AS1 knockdown showed opposite effect on OS-732 cells. In both in vitro and in vivo model, NNT-AS1 expression level significantly correlated with that of beta-catenin, RUNX2, IGF-1R, c-Myc, Cyclin D1 and MMP13 as well as Akt phosphorylation level, and inversely correlated with miR-320a expression. Transfection of miR-320a mimic significantly inhibiter the promoting effect of NNT-AS1 on cell proliferation, survival and mobility of U2OS cells, while miR-320 inhibitor partially rescued that of OS-732 cells. CONCLUSION: NNT-As1 functions as a cancer-promoting lncRNA by downregulating miR-320a, thus increasing the protein expression level of beta-catenin, RUNX2 and IGF-1R as well as activation of Akt in osteosarcoma.
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Neoplasias Óseas/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , NADP Transhidrogenasa AB-Específica/genética , Osteosarcoma/patología , ARN sin Sentido/genética , ARN Largo no Codificante/genética , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Proliferación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Ratones , Ratones Desnudos , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , NADP Transhidrogenasa AB-Específica/antagonistas & inhibidores , Osteosarcoma/genética , Osteosarcoma/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Among mitochondrial NADP-reducing enzymes, nicotinamide nucleotide transhydrogenase (NNT) establishes an elevated matrix NADPH/NADP+ by catalyzing the reduction of NADP+ at the expense of NADH oxidation coupled to inward proton translocation across the inner mitochondrial membrane. Here, we characterize NNT activity and mitochondrial redox balance in the brain using a congenic mouse model carrying the mutated Nnt gene from the C57BL/6J strain. The absence of NNT activity resulted in lower total NADPH sources activity in the brain mitochondria of young mice, an effect that was partially compensated in aged mice. Nonsynaptic mitochondria showed higher NNT activity than synaptic mitochondria. In the absence of NNT, an increased release of H2 O2 from mitochondria was observed when the metabolism of respiratory substrates occurred with restricted flux through relevant mitochondrial NADPH sources or when respiratory complex I was inhibited. In accordance, mitochondria from Nnt-/- brains were unable to sustain NADP in its reduced state when energized in the absence of carbon substrates, an effect aggravated after H2 O2 bolus metabolism. These data indicate that the lack of NNT in brain mitochondria impairs peroxide detoxification, but peroxide detoxification can be partially counterbalanced by concurrent NADPH sources depending on substrate availability. Notably, only brain mitochondria from Nnt-/- mice chronically fed a high-fat diet exhibited lower activity of the redox-sensitive aconitase, suggesting that brain mitochondrial redox balance requires NNT under the metabolic stress of a high-fat diet. Overall, the role of NNT in the brain mitochondria redox balance especially comes into play under mitochondrial respiratory defects or high-fat diet.