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OBJECTIVE: Rheumatoid arthritis (RA) is a progressive disease including four stages, where gut microbiome is associated with pathogenesis. We aimed to investigate stage-specific roles of microbial dysbiosis and metabolic disorders in RA. METHODS: We investigated stage-based profiles of faecal metagenome and plasma metabolome of 76 individuals with RA grouped into four stages (stages I-IV) according to 2010 RA classification criteria, 19 individuals with osteroarthritis and 27 healthy individuals. To verify bacterial invasion of joint synovial fluid, 16S rRNA gene sequencing, bacterial isolation and scanning electron microscopy were conducted on another validation cohort of 271 patients from four RA stages. RESULTS: First, depletion of Bacteroides uniformis and Bacteroides plebeius weakened glycosaminoglycan metabolism (p<0.001), continuously hurting articular cartilage across four stages. Second, elevation of Escherichia coli enhanced arginine succinyltransferase pathway in the stage II and stage III (p<0.001), which was correlated with the increase of the rheumatoid factor (p=1.35×10-3) and could induce bone loss. Third, abnormally high levels of methoxyacetic acid (p=1.28×10-8) and cysteine-S-sulfate (p=4.66×10-12) inhibited osteoblasts in the stage II and enhanced osteoclasts in the stage III, respectively, promoting bone erosion. Fourth, continuous increase of gut permeability may induce gut microbial invasion of the joint synovial fluid in the stage IV. CONCLUSIONS: Clinical microbial intervention should consider the RA stage, where microbial dysbiosis and metabolic disorders present distinct patterns and played stage-specific roles. Our work provides a new insight in understanding gut-joint axis from a perspective of stages, which opens up new avenues for RA prognosis and therapy.
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Wilson's disease (WD) is a rare autosomal recessive disorder of copper metabolism caused by an ATP7B gene mutation. Except for hepatic, neurological symptoms, lower bone mineral density is another most frequent clinical features of WD, but the underlying mechanisms have not been fully understood. This article aims to use induced pluripotent stem cells (iPSCs) to establish cellular osteoblasts model related to WD to identify abnormal osteogenesis and signaling pathways. In this study, we successfully produced functional osteoblasts from normal and WD iPSCs through embryoid bodies (EBs) formation method, and then we found WD osteoblasts may have a lower osteogenesis activity than normal controls by detection of osteogenic marker genes and mineralization ability. Further, through gene expression profiling, detection of ß-catenin in total protein and nuclear protein, and the nuclear localization of ß-catenin, we identified and validated that low osteogenic activity in WD may be due to abnormal ß-catenin pathway. Interestingly, we found SKL2001, a small molecule can reverse decreased osteogenesis of WD. In summery, our results suggested that the low bone density of WD may caused by abnormal ß-catenin signaling pathway, and these may provided a new target for the treatment of WD.
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Degeneração Hepatolenticular/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Osteogênese , Via de Sinalização Wnt , beta Catenina/metabolismo , Células Cultivadas , Degeneração Hepatolenticular/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologiaRESUMO
OBJECTIVE: Tumour necrosis factor alpha (TNF-α) signalling plays a central role in the pathogenesis of various autoimmune diseases, particularly inflammatory arthritis. This study aimed to repurpose clinically approved drugs as potential inhibitors of TNF-α signalling in treatment of inflammatory arthritis. METHODS: In vitro and in vivo screening of an Food and Drug Administration (FDA)-approved drug library; in vitro and in vivo assays for examining the blockade of TNF actions by fexofenadine: assays for defining the anti-inflammatory activity of fexofenadine using TNF-α transgenic (TNF-tg) mice and collagen-induced arthritis in DBA/1 mice. Identification and characterisation of the binding of fexofenadine to cytosolic phospholipase A2 (cPLA2) using drug affinity responsive target stability assay, proteomics, cellular thermal shift assay, information field dynamics and molecular dynamics; various assays for examining fexofenadine inhibition of cPLA2 as well as the dependence of fexofenadine's anti-TNF activity on cPLA2. RESULTS: Serial screenings of a library composed of FDA-approved drugs led to the identification of fexofenadine as an inhibitor of TNF-α signalling. Fexofenadine potently inhibited TNF/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) signalling in vitro and in vivo, and ameliorated disease symptoms in inflammatory arthritis models. cPLA2 was isolated as a novel target of fexofenadine. Fexofenadine blocked TNF-stimulated cPLA2 activity and arachidonic acid production through binding to catalytic domain 2 of cPLA2 and inhibition of its phosphorylation on Ser-505. Further, deletion of cPLA2 abolished fexofenadine's anti-TNF activity. CONCLUSION: Collectively, these findings not only provide new insights into the understanding of fexofenadine action and underlying mechanisms but also provide new therapeutic interventions for various TNF-α and cPLA2-associated pathologies and conditions, particularly inflammatory rheumatic diseases.
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Artrite Experimental/tratamento farmacológico , Fosfolipases A2 Citosólicas/efeitos dos fármacos , Terfenadina/análogos & derivados , Inibidores do Fator de Necrose Tumoral/farmacologia , Animais , Camundongos , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Transdução de Sinais/efeitos dos fármacos , Terfenadina/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Icariin (ICA) is the main active flavonoid glucoside from herbs of the genus Epimedium; in traditional Chinese medicine, these herbs have long been prescribed for the treatment of bone fractures and osteoporosis. Several studies have shown that treatment with ICA can increase osteogenic differentiation and reduce bone loss in vivo and in vitro. However, the definite signaling pathway of this osteogenic effect remains unclear. In this study, we selected bone morphogenetic protein 2 (BMP2)-induced osteoblastic differentiation of multipotent mesenchymal progenitor C2C12 cells as a model of osteoblast differentiation. We investigated the effects of ICA on C2C12 cells osteogenic differentiation and the underlying molecular mechanisms. We found that ICA could enhance BMP2-mediated osteoblastic differentiation of C2C12 cells in a dose-dependent manner. Treatment with ICA activated the cAMP/PKA/CREB signaling axis in a time-dependent manner. Blocking cAMP signaling using the PKA selective inhibitor H89 significantly inhibited the stimulatory effect of ICA on osteogenesis. Therefore, the osteoinductive potential and the low cost of ICA indicate that it is a promising alternative treatment or promoter for enhancing the therapeutic effects of BMP2.
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Proteína Morfogenética Óssea 2/farmacologia , Reabsorção Óssea/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Flavonoides/farmacologia , Osteogênese/efeitos dos fármacos , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , AMP Cíclico/genética , Epimedium/química , Fraturas Ósseas/tratamento farmacológico , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Medicina Tradicional Chinesa , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteogênese/genética , Osteoporose/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacosRESUMO
Many regulators have been identified to participate in the cross-talk between muscle and bone, however, most previous studies focus on secreting proteins. In this study, we demonstrated that exosomes from myoblasts C2C12 can promote pre-osteoblasts MC3T3-E1 differentiation to osteoblasts. We revealed that the effect of C2C12 exosomes depended on its miR-27a-3p component, they can increase miR-27a-3p level in the recipient cells, and decrease its direct target adenomatous polyposis coli (APC) expression, thus activating ß-catenin pathway. Furthermore, C2C12 exosomes failed to exert above effects when miR-27a-3p was deprived. These findings indicates exosomal microRNAs can be regarded as a novel type of "myokines" with osteogenesis promoting potential, which would broad our understanding of the muscle-bone interaction under physiological and pathological conditions.
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Diferenciação Celular/genética , Exossomos/metabolismo , MicroRNAs/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Osteoblastos/citologia , Osteogênese/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Camundongos , MicroRNAs/genética , Osteoblastos/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/metabolismoRESUMO
The aim of this study is to investigate the association of the polymorphisms in tumor necrosis factor (TNF) and granulin (GRN) with ankylosing spondylitis (AS) in a Chinese Han population. Five single nucleotide polymorphisms (SNPs) covering TNF and six SNPs covering GRN were investigated in 861 Chinese Han AS patients and 864 healthy controls. For rs1799964, the C allele was linked to reduced risk of AS (p < 0.0001, OR = 0.60, 95% CI = 0.50-0.71). The carriers of the C/C homozygote showed a significantly lower risk of AS compared with the TT homozygote and the C/T heterozygote under the recessive model (p < 0.0001, OR = 0.23, 95% CI = 0.12-0.45). For rs1800629, the A allele was also linked to reduced risk of AS (p < 0.0001, OR = 0.54, 95% CI = 0.39-0.74). For rs1800630, the A allele was also linked to reduced risk of AS (p < 0.0001, OR = 0.59, 95% CI = 0.48-0.72). The carriers of the A/A homozygote showed a significantly lower risk of AS compared with the C/C homozygote and the A/C heterozygote under the recessive model (p < 0.0001, OR = 0.18, 95% CI = 0.07-0.47). For rs769178, the T allele was linked to increased risk of AS (p < 0.0001, OR = 2.59, 95% CI = 2.18-3.09). The carriers of the T/T homozygote showed a significantly higher risk of AS compared with the GG homozygote and the G/T heterozygote under the recessive model (p < 0.0001, OR = 3.34, 95 %CI = 1.95-5.72). There was no significant difference between the AS patients and the controls in the genotype or allele frequencies of rs361525. For GRN, there was no significant difference between the AS patients and the controls in the genotype or allele frequencies of rs25646, rs3760365, rs3785817, rs4792939, rs5848, rs850713 (p > 0.05). This study indicates that polymorphisms in TNF are related to AS, but polymorphisms in GRN are not related to AS susceptibility in a Chinese Han population.
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Peptídeos e Proteínas de Sinalização Intercelular/genética , Polimorfismo de Nucleotídeo Único , Espondilite Anquilosante/genética , Fator de Necrose Tumoral alfa/genética , Adulto , Povo Asiático/genética , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , China/epidemiologia , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Haplótipos , Heterozigoto , Homozigoto , Humanos , Desequilíbrio de Ligação , Masculino , Razão de Chances , Fenótipo , Progranulinas , Fatores de Risco , Espondilite Anquilosante/diagnóstico , Espondilite Anquilosante/etnologia , Adulto JovemRESUMO
Isopsoralen (IPRN), one of the main effective ingredients in Psoralea corylifolia Linn, has a variety of biological effects, including antiosteoporotic effects. In vivo studies show that IPRN can increase bone strength and trabecular bone microstructure in a sex hormone deficiency-induced osteoporosis model. However, the mechanism underlying this osteogenic potential has not been investigated in detail. In the present study, we investigated the molecular mechanism of IPRN-induced osteogenesis in MC3T3-E1 cells. Isopsoralen promoted osteoblast differentiation and mineralization, increased calcium nodule levels and alkaline phosphatase (ALP) activity and upregulated osteoblast markers, including ALP, runt-related transcription factor 2 (RUNX2), and collagen type I alpha 1 chain (COL1A1). Furthermore, IPRN limited the nucleocytoplasmic shuttling of aryl hydrocarbon receptor (AhR) by directly binding to AhR. The AhR target gene cytochrome P450 family 1 subfamily A member 1 (CYP1A1) was also inhibited in vitro and in vivo. This effect was inhibited by the AhR agonists indole-3-carbinol (I3C) and 3-methylcholanthrene (3MC). Moreover, IPRN also increased estrogen receptor alpha (ERα) expression in an AhR-dependent manner. Taken together, these results suggest that IPRN acts as an AhR antagonist and promotes osteoblast differentiation via the AhR/ERα axis.
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Receptor alfa de Estrogênio/metabolismo , Furocumarinas/farmacologia , Osteogênese/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Receptor alfa de Estrogênio/genética , Furocumarinas/química , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Estrutura Molecular , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Ligação Proteica , Transporte Proteico , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
MicroRNAs have emerged as pivotal regulators in various physiological and pathological processes, including osteogenesis. Here we discuss the contribution of miR-5100 to osteoblast differentiation and mineralization. We found that miR-5100 was upregulated during osteoblast differentiation in ST2 and MC3T3-E1 cells. Next, we verified that miR-5100 can promote osteogenic differentiation with gain-of-function and loss-of-function experiments. Target prediction analysis and experimental validation demonstrated that Tob2, which acts as a negative regulator of osteogenesis, was negatively regulated by miR-5100. Furthermore, we confirmed that the important bone-related transcription factor osterix, which can be degraded by binding to Tob2, was influenced by miR-5100 during osteoblast differentiation. Collectively, our results revealed a new molecular mechanism that fine-tunes osteoblast differentiation through miR-5100/Tob2/osterix networks.
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Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , MicroRNAs/metabolismo , Osteogênese/genética , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Camundongos , MicroRNAs/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismoRESUMO
Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that ß-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit the level of connexin43, a key regulator of osteoblast differentiation by affecting ß-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating ß-catenin/connexin43 pathway and osteoblast differentiation.
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Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Células 3T3 , Animais , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , CamundongosRESUMO
Membrane proteins are an important source of potential targets for anticancer drugs or biomarkers for early diagnosis. In this study, we used a modified aqueous two-phase partition system combined with two-dimensional (2D) matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS, 2D-MALDI-TOF-TOF-MS/MS) analysis to isolate and identify membrane proteins in PANC-1 pancreatic cancer cells. Using this method, we identified 55 proteins, of which 31 (56.4 %) were membrane proteins, which, according to gene ontology annotation, are associated with various cellular processes including cell signal transduction, differentiation, and apoptosis. Immunohistochemical analysis showed that the expression level of one of the identified mitochondria membrane proteins, prohibitin 1 (PHB1), is correlated with pancreatic carcinoma differentiation; PHB1 is expressed at a higher level in normal pancreatic tissue than in well-differentiated carcinoma tissue. Further studies showed that PHB1 plays a proapoptotic role in human pancreatic cancer cells, which suggests that PHB1 has antitumorigenic properties. In conclusion, we have provided a modified method for isolating and identifying membrane proteins and demonstrated that PHB1 may be a promising biomarker for early diagnosis and therapy of pancreatic (and potentially other) cancers.
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Biomarcadores Tumorais/biossíntese , Proteínas de Membrana/biossíntese , Neoplasias Pancreáticas/genética , Proteínas Repressoras/biossíntese , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Diferenciação Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/isolamento & purificação , Neoplasias Pancreáticas/tratamento farmacológico , Prognóstico , Proibitinas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Neoplasias PancreáticasRESUMO
OBJECTIVES: Docetaxel was the first drug with proven survival benefit in men with castration-resistant prostate cancer. Acquired resistance to docetaxel precedes fatality in castration-resistant prostate cancer. The aims of this study were to evaluate docetaxel-sensitive and docetaxel-resistant proteomes in PC-3 cells, and to investigate the molecular mechanism of docetaxel-resistant PC-3 cells. METHODS: Docetaxel-resistant PC-3 cells were developed by docetaxel dose escalation. The global profiling of the protein expression was investigated in docetaxel-sensitive and docetaxel-resistant proteomes in PC-3 cells using 2-dimensional polyacrylamide gel electrophoresis/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: Forty-nine differential proteins were found in docetaxel-resistant PC-3 cells in comparison with docetaxel-sensitive PC-3 cells. Expression in 29 proteins was upregulated, whereas expression in 20 proteins was downregulated. ATP synthase and galectin-1 were involved in the formation of tumor vessels; calreticulin, cathepsin D, and cofilin were involved in tumor metastasis, and GRP78 (78-kDa glucose-regulated protein) and microtubule-associated protein-6 were involved in drug resistance of tumor. CONCLUSION: It is suggested that a proteomic expression difference exists between docetaxel-sensitive and docetaxel-resistant PC-3 cells, which would be helpful for further understanding the molecular mechanisms of docetaxel resistance in PC-3 cells.
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Antineoplásicos/química , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Taxoides/química , Antineoplásicos/uso terapêutico , Calreticulina/metabolismo , Catepsina D/metabolismo , Linhagem Celular Tumoral , Cofilina 1/metabolismo , Docetaxel , Eletroforese em Gel Bidimensional , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Humanos , Concentração Inibidora 50 , Masculino , Espectrometria de Massas , Metástase Neoplásica , Neoplasias de Próstata Resistentes à Castração/metabolismo , Proteoma , Proteômica , Taxoides/uso terapêutico , Regulação para CimaRESUMO
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic and symmetric in-flammation. Our previous research revealed an imbalance in the gut flora of RA patients and showed that certain gut microbiota can accelerate RA progression by enhancing vitamin C degradation. However, it is unclear whether vitamin C supplementation could improve the gut microbiota to prevent the development of arthritis by interfering with the gut-joint axis. In this work, we aimed to evaluate the effects of vitamin C in regulating the gut microbiota and to elucidate its potential role in the onset and progression of RA in a mouse model, thus providing a basis for the development of new intervention strategies and treatments for RA. In this study, collagen-induced arthritis (CIA) mouse models, biochemical, histological and 16S rRNA microbiological methods were used to investigate the role and possible mechanism of vitamin C in rheumatoid arthritis. The results showed that treatment of CIA mice with vitamin C effectively rescued the gut mi-crobiota imbalance and suppressed the inflammatory response associated with RA, and effectively alleviated arthritis symptoms in mice in which levels of the pro-inflammatory cytokines IL-6 and TNF-α were specifi-cally reduced. In conclusion, our results demonstrate the potential of vitamin C as a potential therapeutic choice for RA.
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Artrite Experimental , Artrite Reumatoide , Ácido Ascórbico , Microbioma Gastrointestinal , Animais , Ácido Ascórbico/uso terapêutico , Ácido Ascórbico/administração & dosagem , Ácido Ascórbico/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/microbiologia , Camundongos , Artrite Experimental/tratamento farmacológico , Artrite Experimental/microbiologia , Artrite Experimental/imunologia , Masculino , Camundongos Endogâmicos DBA , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Modelos Animais de Doenças , RNA Ribossômico 16S/genéticaRESUMO
Intervertebral disc is a highly rhythmical tissue. As a key factor linking biorhythm and inflammatory response, the shielding effect of NR1D1 in the process of intervertebral disc degeneration remains unclear. Here, we first confirmed that NR1D1 in the nucleus pulposus tissue presents periodic rhythmic changes and decreases in expression with intervertebral disc degeneration. Second, when NR1D1 was activated by SR9009 in vitro, NLRP3 inflammasome assembly and IL-1ß production were inhibited, while ECM synthesis was increased. Finally, the vivo experiments further confirmed that the activation of NR1D1 can delay the process of disc degeneration to a certain extent. Mechanistically, we demonstrate that NR1D1 can bind to IL-1ß and NLRP3 promoters, and that the NR1D1/NLRP3/IL-1ß pathway is involved in this process. Our results demonstrate that the activation of NR1D1 can effectively reduce IL-1ß secretion, alleviate LPS-induced NPMSC pyroptosis, and protect ECM degeneration.
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Human hematopoiesis starts at early yolk sac and undergoes site- and stage-specific changes over development. The intrinsic mechanism underlying property changes in hematopoiesis ontogeny remains poorly understood. Here, we analyzed single-cell transcriptome of human primary hematopoietic stem/progenitor cells (HSPCs) at different developmental stages, including yolk-sac (YS), AGM, fetal liver (FL), umbilical cord blood (UCB) and adult peripheral blood (PB) mobilized HSPCs. These stage-specific HSPCs display differential intrinsic properties, such as metabolism, self-renewal, differentiating potentialities etc. We then generated highly co-related gene regulatory network (GRNs) modules underlying the differential HSC key properties. Particularly, we identified GRNs and key regulators controlling lymphoid potentiality, self-renewal as well as aerobic respiration in human HSCs. Introducing selected regulators promotes key HSC functions in HSPCs derived from human pluripotent stem cells. Therefore, GRNs underlying key intrinsic properties of human HSCs provide a valuable guide to generate fully functional HSCs in vitro.
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Emergency myelopoiesis (EM) is essential in immune defense against pathogens for rapid replenishing of mature myeloid cells. During the EM process, a rapid cell-cycle switch from the quiescent hematopoietic stem cells (HSCs) to highly proliferative myeloid progenitors (MPs) is critical. How the rapid proliferation of MPs during EM is regulated remains poorly understood. Here, we reveal that ATG7, a critical autophagy factor, is essential for the rapid proliferation of MPs during human myelopoiesis. Peripheral blood (PB)-mobilized hematopoietic stem/progenitor cells (HSPCs) with ATG7 knockdown or HSPCs derived from ATG7-/- human embryonic stem cells (hESCs) exhibit severe defect in proliferation during fate transition from HSPCs to MPs. Mechanistically, we show that ATG7 deficiency reduces p53 localization in lysosome for a potential autophagy-mediated degradation. Together, we reveal a previously unrecognized role of autophagy to regulate p53 for a rapid proliferation of MPs in human myelopoiesis.
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Mielopoese , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células Mieloides , Autofagia/genéticaRESUMO
MicroRNAs (miRNAs) are a class of small endogenous gene regulators that have been implicated in various developmental and pathological processes. However, the precise identities and functions of miRNAs involved in antitumor immunity are not yet well understood. miRNA-21 is an oncogenic miRNA that can be detected in various tumours. In this study, we report that a miRNA-21 inhibitor enhances the release of chemoattractants RANTES and IP-10 in the MCF-7 breast cancer cell line and results in increased lymphocyte migration. Thus, miRNA-21 is a potential therapeutic target for cancer immunotherapy. We further demonstrated that PIAS3, a protein inhibitor of activated STAT3, is a target of miRNA-21 in MCF-7. Thus, miRNA-21 is a novel miRNA regulating immune cell recruitment, which acts at least in part via its inhibition of PIAS3 expression and oncogenic STAT3 signalling in tumour cells.
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Neoplasias da Mama/imunologia , Quimiocina CCL5/imunologia , Quimiocina CXCL10/imunologia , MicroRNAs/imunologia , Chaperonas Moleculares/imunologia , Proteínas Inibidoras de STAT Ativados/imunologia , Fator de Transcrição STAT3/imunologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Linfócitos/imunologia , Linfócitos/metabolismo , MicroRNAs/genética , Chaperonas Moleculares/genética , Proteínas Inibidoras de STAT Ativados/genética , Interferência de RNA , Transdução de SinaisRESUMO
BACKGROUND: The conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins. Studies have found PADI4, an enzyme performing citrullination, to be highly expressed in a variety of malignant tumours and have shown that PADI4 participates in the process of tumorigenesis. However, as citrullinated proteins have not been systematically investigated in tumours, the present study aimed to identify novel citrullinated proteins in tumours by 2-D western blotting (2-D WB). METHODS: Two identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ECA, H292, HeLa, HEPG2, Lovo, MCF-7, PANC-1, SGC, and SKOV3 tumour cell lines. The expression profiles on a 2-DE gel were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody. By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry. Immunoprecipitation was used to verify the expression and citrullination of the targeted proteins in tumour cell lines. RESULTS: 2-D WB and mass spectrometry identified citrullinated α-enolase (ENO1), heat shock protein 60 (HSP60), keratin 8 (KRT8), tubulin beta (TUBB), T cell receptor chain and vimentin in these cell lines. Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumour cell lines. CONCLUSIONS: The citrullination of these proteins suggests a new mechanism in the tumorigenic process.
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Biomarcadores Tumorais/metabolismo , Citrulina/metabolismo , Neoplasias/metabolismo , Western Blotting , Humanos , Imunoprecipitação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais Cultivadas , Eletroforese em Gel Diferencial BidimensionalRESUMO
OBJECTIVE: Cuproptosis-related genes are closely related to lung adenocarcinoma (LUAD), which can be analyzed via the analysis of long noncoding RNA (lncRNA). To date, the clinical significance and function of cuproptosis-related lncRNAs are still not well elucidated. Further analysis of cuproptosis-related prognostic lncRNAs is of great significance for the treatment, diagnosis, and prognosis of LUAD. METHODS: In this study, a multiple machine learning (ML)-based computational approach was proposed for the identification of the cuproptosis-related lncRNAs signature (CRlncSig) via comprehensive analysis of cuproptosis, lncRNAs, and clinical characteristics. The proposed approach integrated multiple ML algorithms (least absolute shrinkage and selection operator regression analysis, univariate and multivariate Cox regression) to effectively identify the CRlncSig. RESULTS: Based on the proposed approach, the CRlncSig was identified from the 3450 cuproptosis-related lncRNAs, which consist of 13 lncRNAs (CDKN2A-DT, FAM66C, FAM83A-AS1, AL359232.1, FRMD6-AS1, AC027237.4, AC023090.1, AL157888.1, AL627443.3, AC026355.2, AC008957.1, AP000346.1, and GLIS2-AS1). CONCLUSIONS: The CRlncSig could well predict the prognosis of different LUAD patients, which is different from other clinical features. Moreover, the CRlncSig was proved to be an effective indicator of patient survival via functional characterization analysis, which is relevant to cancer progression and immune infiltration. Furthermore, the results of RT-PCR assay indicated that the expression level of FAM83A-AS1 and AC026355.2 in A549 and H1975 cells (LUAD) was significantly higher than that in BEAS-2B cells (normal lung epithelial).
Assuntos
RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , Prognóstico , Imunoterapia , Relevância Clínica , Aprendizado de Máquina , Apoptose , Proteínas de NeoplasiasRESUMO
OBJECTIVE: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a rare acquired immune-mediated neuropathy. Although microbial infection is potentially a contributing factor, a causative link between CIDP and microbial infection remains unclear. There is also no definitive biomarker for CIDP diagnostics and therapies. The present study aimed to characterize the serum metabolic profile and gut microbiome structure in CIDP. METHODS: Targeted metabolomics profiling of serum, using liquid chromatography-mass spectrometry, and metagenomics sequencing of stool samples from a cohort of CIDP and non-CIDP subjects were performed to evaluate serum metabolic profiles and gut microbiome structure in CIDP subjects relative to healthy controls. RESULTS: Metabolome data revealed that the bile acids profile was perturbed in CIDP with bile acids and arachidonic acid enriched significantly in CIDP versus non-CIDP controls. Metagenome data revealed that opportunistic pathogens, such as Klebsiella pneumonia and Megamonas funiformis, and genes involved in bacterial infection were notably more abundant in CIDP subjects, while gut microbes related to biotransformation of secondary bile acids were abnormal in CIDP versus non-CIDP subjects. Correlation analysis revealed that changes in secondary bile acids were associated with altered gut microbes, including Bacteroides ovatus, Bacteroides caccae, and Ruminococcus gnavus. CONCLUSION: Bile acids and arachidonic acid metabolism were disturbed in CIDP subjects and might be affected by the dysbiosis of gut microbial flora. These findings suggest that the combination of bile acids and arachidonic acid could be used as a CIDP biomarker and that modulation of gut microbiota might impact the clinical course of CIDP.
RESUMO
Methylmalonic acidemia with homocystinuria (MMA-cblC) is an autosomal recessive genetic disorder of organic acid metabolism. Shandong, a northern province of China, has a significantly high incidence of about 1/4,000, suggesting a high carrying rate among the local population. The current study established a PCR technique involving high-resolution melting (HRM) to screen for carriers based on hotspot mutation analysis to further develop a preventive strategy to reduce the local incidence of this rare disease. Whole-exome sequencing of 22 families with MMA-cblC and a comprehensive literature review were used to identify MMACHC hotspot mutations in Shandong Province. Subsequently, a PCR-HRM assay based on the selected mutations was established and optimized for large-scale hotspot mutation screening. The accuracy and efficiency of the screening technique was validated using samples from 69 individuals with MMA-cblC and 1,000 healthy volunteers. Six hotspot mutations in the MMACHC gene (c.609G>A, c.658_660delAAG, c.80A>G, c.217C>T, c.567dupT and c.482G>A), which account for 74% of the alleles associated with MMA-cblC, were used to establish a screening technique. The established PCR-HRM assay detected 88 MMACHC mutation alleles in a validation study with 100% accuracy. In the general population in Shandong, the carrying rate of 6 MMACHC hotspot mutations was 3.4%. In conclusion, the 6 hotspots identified cover the majority of the MMACHC mutation spectrum, and the Shandong population has a particularly high carrying rate of MMACHC mutations. The PCR-HRM assay is highly accurate, cost-effective, and easy to use, making it an ideal choice for mass carrier screening.