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2.
Life Sci ; 232: 116630, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31279783

RESUMO

AIMS: Lung adenocarcinoma consists of multiple therapeutic targets, however, patients will inevitably progress to later stage diagnosis with Tyrosine Kinase Inhibitor treatment resistance. We aim to investigate the roles of non-coding TUSC7 in ordering the cell division tendency, helping to sensitize the resistance in a miRNA incorporating way. MATERIALS AND METHODS: Online study of bioinformatics analysis, molecular experiments of luciferase test, immunofluorescence staining and qRT-PCR were applied to dig out the mechanistic regulations. KEY FINDINGS: TUSC-7 inhibited the renewal ability of adenocarcinoma stem cells, yielding to asymmetric cell splitting. Informatics analysis and the luciferase testing confirmed the 3'UTR binding site, and revealed the post-transcriptional regulation of NUMB referring to miR-146. TUSC-7 sponged miR-146 and abolished its degradation toward to NUMB, and this integrated cascade made several genes become tangled to full functionality. SIGNIFICANCE: TUSC-7 was proved to be one strong suppressive lnc-RNA in lung adenocarcinoma stem cells, functioning through inactivating NOTCH signaling, and the turbulence on division modes precisely pointed to the key mechanisms of stem cells' renewal. The decreasing of tumor suppressive miR-146 was necessary in TUSC-7 conducted renewal repression, despite it alone could also reduce the renewal efficiency, indicating that more complicated non-coding genes may be involved in its regulation.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/fisiologia , Regiões 3' não Traduzidas , Adenocarcinoma de Pulmão/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo
3.
DNA Cell Biol ; 38(7): 688-699, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31188028

RESUMO

This study was aimed to identify hub genes associated with the development of glioblastoma (GBM) by conducting a bioinformatic analysis. The raw gene expression data were downloaded from the Gene Expression Omnibus database and The Cancer Genome Atlas project. After the differentially expressed genes (DEGs) were identified, the functional enrichment analysis of DEGs was conducted. Subsequently, the protein-protein interaction (PPI) network, molecular complex detection clusters, and transcriptional factor (TF)-miRNA-target regulatory network were constructed, respectively. Furthermore, the survival analysis of prognostic outcomes and genes was analyzed. In addition, the expression of key genes was validated by quantitative real-time PCR (qRT-PCR) analysis. A total of 884 DEGs, including 418 upregulated and downregulated genes, were identified between GBM and normal samples. The PPI network comprised a set of 3418 pairs involving 751 nodes, and AKT1 and CDK2 were the critical genes in the network. A total of seven clusters were identified, the genes in which were intensively associated with cell cycle, cholinergic synapse, and extracellular matrix (ECM)-receptor interaction. qRT-PCR analysis indicated that AKT1 and CDK2 were significantly upregulated, and NRXN3 and NPTX2 were significantly downregulated in GBM samples. The TF-miRNA-target regulatory networks were built, in which CCNB1, RFC5, microRNA524, and microRNA34b were key regulators. There were 43 genes, including NPTX2 and NRXN3, significantly related to the prognostic outcomes of GBM patients. These crucial genes might be promising options for GBM treatment.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , MicroRNAs/genética , Transcriptoma , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Células Cultivadas , Ciclina B1/genética , Ciclina B1/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo
4.
Nat Commun ; 10(1): 2612, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197141

RESUMO

Primary microcephaly is caused by mutations in genes encoding centrosomal proteins including WDR62 and KIF2A. However, mechanisms underlying human microcephaly remain elusive. By creating mutant mice and human cerebral organoids, here we found that WDR62 deletion resulted in a reduction in the size of mouse brains and organoids due to the disruption of neural progenitor cells (NPCs), including outer radial glia (oRG). WDR62 ablation led to retarded cilium disassembly, long cilium, and delayed cell cycle progression leading to decreased proliferation and premature differentiation of NPCs. Mechanistically, WDR62 interacts with and promotes CEP170's localization to the basal body of primary cilium, where CEP170 recruits microtubule-depolymerizing factor KIF2A to disassemble cilium. WDR62 depletion reduced KIF2A's basal body localization, and enhanced KIF2A expression partially rescued deficits in cilium length and NPC proliferation. Thus, modeling microcephaly with cerebral organoids and mice reveals a WDR62-CEP170-KIF2A pathway promoting cilium disassembly, disruption of which contributes to microcephaly.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesina/metabolismo , Microcefalia/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Cílios/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/patologia , Neuroglia/citologia , Neuroglia/patologia , Organoides/patologia , Fosfoproteínas/genética , RNA Interferente Pequeno/metabolismo
5.
Gastroenterology ; 157(3): 838-850.e6, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31163177

RESUMO

BACKGROUND & AIMS: Little is known about mechanisms of perineural invasion (PNI) by pancreatic ductal adenocarcinomas (PDAs) or other tumors. Annexin A2 (ANXA2) regulates secretion of SEMA3D, an axon guidance molecule, which binds and activates the receptor PLXND1 to promote PDA invasion and metastasis. We investigated whether axon guidance molecules promote PNI and metastasis by PDA cells in mice. METHODS: We performed studies in a dorsal root ganglion (DRG) invasion system, wild-type C57BL/6 mice (controls), mice with peripheral sensory neuron-specific disruption of PlxnD1 (PLAC mice), LSL-KRASG12D/+;LSL-TP53R172H/+;PDX-1-CRE+/+ (KPC) mice, and KPC mice crossed with ANXA2-knockout mice (KPCA mice). PDA cells were isolated from KPC mice and DRG cells were isolated from control mice. Levels of SEMA3D or ANXA2 were knocked down in PDA cells with small hairpin and interfering RNAs and cells were analyzed by immunoblots in migration assays, with DRGs and with or without antibodies against PLXND1. PDA cells were injected into the pancreas of control and PLAC mice, growth of tumors was assessed, and tumor samples were analyzed by histology. DRG cells were incubated with SEMA3D and analyzed by live imaging. We measured levels of SEMA3D and PLXND1 in PDA specimens from patients with PNI and calculated distances between tumor cells and nerves. RESULTS: DRG cells increase the migration of PDC cells in invasion assays; knockdown of SEMA3D in PDA cells or antibody blockade of PLXND1 on DRG cells reduced this invasive activity. In mice, orthotopic tumors grown from PDA cells with knockdown of SEMA3D, and in PLAC mice, orthotopic tumors grown from PDA cells, had reduced innervation and formed fewer metastases than orthotopic tumors grown from PDA cells in control mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. CONCLUSIONS: DRG cells increase the migratory and invasive activities of pancreatic cancer cells, via secretion of SEMA3D by pancreatic cells and activation of PLXND1 on DRGs. Knockdown of SEMA3D and loss of neural PLXND1 reduces innervation of orthotopic PDAs and metastasis in mice. Increased levels of SEMA3D and PLXND1 in human PDA specimens associated with PNI. Strategies to disrupt the axon guidance pathway mediated by SEMA3D and PLXND1 might be developed to slow progression of PDA.


Assuntos
Anexina A2/metabolismo , Orientação de Axônios , Carcinoma Ductal Pancreático/metabolismo , Movimento Celular , Gânglios Espinais/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Pancreáticas/metabolismo , Semaforinas/metabolismo , Animais , Anexina A2/deficiência , Anexina A2/genética , Orientação de Axônios/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/secundário , Comunicação Celular , Gânglios Espinais/patologia , Regulação Neoplásica da Expressão Gênica , Genes p53 , Genes ras , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Humanos , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Invasividade Neoplásica , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Crescimento Neuronal , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fenótipo , Semaforinas/genética , Transdução de Sinais , Transativadores/genética , Células Tumorais Cultivadas
6.
Nat Commun ; 10(1): 2477, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31171770

RESUMO

Blood vessels in the central nervous system (CNS) develop unique features, but the contribution of CNS neurons to regulating those features is not fully understood. We report that inhibiting spontaneous cholinergic activity or reducing starburst amacrine cell numbers prevents invasion of endothelial cells into the deep layers of the retina and causes blood-retinal-barrier (BRB) dysfunction in mice. Vascular endothelial growth factor (VEGF), which drives angiogenesis, and Norrin, a Wnt ligand that induces BRB properties, are decreased after activity blockade. Exogenous VEGF restores vessel growth but not BRB function, whereas stabilizing beta-catenin in endothelial cells rescues BRB dysfunction but not vessel formation. We further identify that inhibiting cholinergic activity reduces angiogenesis during oxygen-induced retinopathy. Our findings demonstrate that neural activity lies upstream of VEGF and Norrin, coordinating angiogenesis and BRB formation. Neural activity originating from specific neural circuits may be a general mechanism for driving regional angiogenesis and barrier formation across CNS development.


Assuntos
Células Amácrinas/fisiologia , Barreira Hematorretiniana/crescimento & desenvolvimento , Neurônios Colinérgicos/fisiologia , Células Endoteliais/fisiologia , Neovascularização Fisiológica/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/inervação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Neurônios Colinérgicos/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proteínas do Olho/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Agonistas Nicotínicos/farmacologia , Oxigênio/efeitos adversos , Piridinas/farmacologia , Doenças Retinianas , Células Ganglionares da Retina/metabolismo , Neovascularização Retiniana/etiologia , Tetrodotoxina/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/metabolismo
7.
Nat Commun ; 10(1): 2549, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186409

RESUMO

Human adipose tissue (hAT) is constituted of structural units termed lobules, the organization of which remains to be defined. Here we report that lobules are composed of two extracellular matrix compartments, i.e., septa and stroma, delineating niches of CD45-/CD34+/CD31- progenitor subsets characterized by MSCA1 (ALPL) and CD271 (NGFR) expression. MSCA1+ adipogenic subset is enriched in stroma while septa contains mainly MSCA1-/CD271- and MSCA1-/CD271high progenitors. CD271 marks myofibroblast precursors and NGF ligand activation is a molecular relay of TGFß-induced myofibroblast conversion. In human subcutaneous (SC) and visceral (VS) AT, the progenitor subset repartition is different, modulated by obesity and in favor of adipocyte and myofibroblast fate, respectively. Lobules exhibit depot-specific architecture with marked fibrous septa containing mesothelial-like progenitor cells in VSAT. Thus, the human AT lobule organization in specific progenitor subset domains defines the fat depot intrinsic capacity to remodel and may contribute to obesity-associated cardiometabolic risks.


Assuntos
Tecido Adiposo/anatomia & histologia , Tecido Adiposo/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Adipócitos/metabolismo , Adipogenia , Fosfatase Alcalina , Diferenciação Celular , Matriz Extracelular , Humanos , Gordura Intra-Abdominal/citologia , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Obesidade , Receptores de Fator de Crescimento Neural/metabolismo , Células-Tronco/metabolismo , Gordura Subcutânea/citologia , Fator de Crescimento Transformador beta1/farmacologia
8.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035716

RESUMO

Konjac ceramide (kCer), which consists of plant-type molecular species of characteristic shingoid bases and fatty acids, is prepared from konjac glucosylceramide GlcCer by chemoenzymatical deglucosylation. kCer activates the semaphorin 3A (Sema3A) signaling pathway, inducing collapsin response mediator protein 2 (CRMP2) phosphorylation. This results in neurite outgrowth inhibition and morphological changes in remaining long neurites in PC12 cells. Whether a specific molecular species of kCer can bind to the Sema3A receptor (Neuropilin1, Nrp1) and activate the Sema3A signaling pathway remains unknown. Here, we prepared kCer molecular species using endoglycoceramidase I-mediated deglucosylation and examined neurite outgrowth and phosphorylation of collapsin response mediator protein 2 in nerve growth factor (NGF)-primed cells. The 8-trans unsaturation of sphingadienine of kCer was essential for Sema3A-like signaling pathway activation. Conversely, 8-cis unsaturation of kCer molecular species had no effect on Sema3A-like activation, and neurite outgrowth inhibition resulted in remaining short neurites. In addition, α-hydroxylation of fatty acids was not associated with the Sema3A-like activity of the kCer molecular species. These results suggest that 8-trans or 8-cis isomerization of sphingadienine determines the specific interactions at the ligand-binding site of Nrp1.


Assuntos
Amorphophallus/química , Etanolaminas/farmacologia , Crescimento Neuronal/efeitos dos fármacos , Animais , Linhagem Celular , Etanolaminas/química , Evolução Molecular , Ácidos Graxos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Semaforina-3A/metabolismo
9.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(5): 508-514, 2019 May 30.
Artigo em Chinês | MEDLINE | ID: mdl-31140412

RESUMO

OBJECTIVE: To investigate the effect of blocking pannexin-1 against acute kidney injury induced by cisplatin. METHODS: Twenty-six male C57BL/6 mice aged 6-8 weeks were randomly divided into control group, cisplatin model (Cis) group and cisplatin + carbenoxolone treatment group (Cis + CBX). In Cis group and Cis + CBX group, the mice were injected intraperitoneally with 20 mg/kg of cisplatin and with CBX (20 mg/kg) at 30 min before and 24 and 48 h after cisplatin inhjection, respectively. All the mice were sacrificed at 72 h after cisplatin injection, and plasma and kidney samples were collected for testing mRNA and protein expression levels of pannexin-1 in the renal tissue using RT-qPCR and Western blotting and for detecting plasma creatinine and BUN levels; the pathological changes in the renal tissues were observed using Periodic Acid-Schiff staining. The expression of kidney injury molecule 1 (KIM-1) was examined using immunohistochemistry and the mRNA expressions of KIM-1 and neutrophil gelatinase- related lipid transport protein (NGAL) were detected by RT-qPCR to evaluate the injuries of the renal tubules. The infiltration of F4/80-positive macrophages and CD4-positive T cells were observed by immunofluorescence. In the in vitro experiment, human proximal tubule epithelial cell line HK-2 was stimulated with 50 µmol/L cisplatin to establish a cell model of acute kidney injury, and the mRNA and protein expressions of pannexin-1 were detected by RT-qPCR and Western blotting at 4, 6, 12, 18 and 24 h after the stimulation. RESULTS: Compared with the control mice, the cisplatin-treated mice showed significantly up-regulated protein levels (P < 0.05) and mRNA levels (P < 0.005) of pannexin-1 in the kidney tissue. Cisplatin stimulation also caused significant increases in the protein levels (P < 0.005) and mRNA levels (P < 0.005) of pannexin-1 in cultured HK-2 cells. Compared with cisplatin-treated mice, the mice treated with both cisplatin and the pannexin-1 inhibitor CBX showed obviously lessened kidney pathologies and milder renal tubular injuries with significantly reduced plasma BUN and Scr levels (P < 0.01), expressions of KIM-1 and NGAL in the kidney (P < 0.05), and infiltration of F4/80-positive macrophages (P < 0.01) and CD4- positive T cells (P < 0.05) in the kidney tissues. CONCLUSIONS: In cisplatin induced acute kidney injury mice model, Pannexin-1 expression is up-regulated in the kidneys tissue, and blocking pannexin-1 alleviates the acute kidney injury via reducing renal inflammatory cell infiltration.


Assuntos
Lesão Renal Aguda , Cisplatino , Conexinas , Reagentes para Ligações Cruzadas , Proteínas do Tecido Nervoso , Lesão Renal Aguda/tratamento farmacológico , Lesão Renal Aguda/metabolismo , Animais , Cisplatino/farmacologia , Conexinas/efeitos dos fármacos , Conexinas/metabolismo , Reagentes para Ligações Cruzadas/farmacologia , Humanos , Rim , Túbulos Renais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Distribuição Aleatória
10.
Dis Markers ; 2019: 6171782, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061682

RESUMO

Renal cancer is one of the most common malignant urological tumors; however, its diagnosis and treatment are not well established. In the present study, we identified that CDK5 regulatory subunit-associated protein 3 (CDK5RAP3), a putative tumor suppressor in many cancers, was downregulated in renal cancer tissues. Through loss- and gain-of-function experiments, we observed that the action of CDK5RAP3 in renal cancer cells was different in Caki-1 and 769-P cell lines. Knockdown of endogenous CDK5RAP3 in Caki-1 slightly increased cell viability, whereas overexpression of CDK5RAP3 in 769-P cells inhibited cell viability. In addition, we observed that CDK5RAP3 participated in the regulation of autophagy in renal cancer. Knockdown of CDK5RAP3 induced significant inhibition of autophagy in Caki-1 cells but not in 769-P cells. In contrast, overexpression of CDK5RAP3 significantly activated autophagy in 769-P cells, as evidenced by increased LC3-II levels. However, the LC3-II could not be altered by CDK5RAP3 overexpression in Caki-1 cells. These findings demonstrated that CDK5RAP3 is downregulated in renal cancer and may be associated with autophagy.


Assuntos
Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Renais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Idoso , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética
11.
Mol Med Rep ; 19(6): 5321-5334, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059045

RESUMO

High pressure is the most important factor inducing retinal ganglion cell (RGC) apoptosis. However, the underlying mechanisms remain obscure. The present study investigated the effects of different levels of hydrostatic pressure (HP) on RGCs and the potential mechanisms involved. Primary cultured rat RGCs were exposed to five levels of HP (0, 20, 40, 60 and 80 mmHg) for 24 h. Morphological changes in RGCs were observed. The viability and apoptosis rate of RGCs were detected using a Cell Counting Kit­8 assay and Annexin V­fluorescein isothiocyanate/propidium iodide flow cytometry, respectively. Western blotting, reverse transcription­quantitative polymerase chain reaction and immunofluorescence were used to detect the expression and mRNA levels of nerve growth factor (NGF), protein kinase B (AKT), apoptosis signal­regulating kinase 1 (ASK1), forkhead box O1 (FoxO1) and cAMP response element binding protein (CREB). In the 0­ and 20­mmHg groups, there were no apoptotic morphological changes. In the 40 mmHg group, parts of the cell were shrunken or disrupted. In the 60 mmHg group, neurite extension was weakened and parts of the cells were disintegrating or dying. In the 80 mmHg group, the internal structures of the cells were not visible at all. The apoptosis rates of RGCs were significantly higher and the viability rates significantly lower under 40, 60 and 80 mmHg compared with under 0 or 20 mmHg (all P<0.01). The expression and mRNA levels of NGF, AKT and CREB decreased in a dose­dependent manner in the 40­, 60­ and 80­mmHg groups (all P<0.05), but those of ASK1 and FoxO1 increased in a dose­dependent manner (all P<0.05). Interestingly, the alterations to the expression and mRNA levels of CREB were significantly larger compared with the changes in ASK1 or FoxO1 in the 40­, 60­ and 80­mmHg groups (all P<0.01). The results of the present study demonstrate that elevated HP of 40, 60 or 80 mmHg reduces viability and induces apoptosis in RGCs, which may occur through effects on the NGF/ASK1/FoxO1 and NGF/AKT/CREB pathways, of which the latter is more strongly affected.


Assuntos
Apoptose , Fator de Crescimento Neural/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Pressão Hidrostática , Fator de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo
12.
Fish Physiol Biochem ; 45(3): 921-933, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31104250

RESUMO

In fish, as in mammals, several studies have demonstrated that the cocaine- and amphetamine-regulated transcript (CART) plays an important role in feeding. However, thus far, the function of CART in gibel carp (Carassius auratus gibelio) feeding regulation has not been reported. In our study, we first identified three forms of CART peptide precursors from gibel carp brain and named these CART-1, CART-2, and CART-3. The full-length cDNA sequences of CART-1, CART-2, and CART-3 were 616 bp, 705 bp, and 760 bp, respectively, encoding peptides of 118, 120, and 104 amino acid residues. We detected mRNA expression of CART-1, CART-2, and CART-3 in a wide range of peripheral and central tissues, with the highest expression detected in the brain. After a meal, mRNA expression of CART-1, CART-2, and CART-3 was significantly elevated, suggesting that CART-1, CART-2, and CART-3 may act as postprandial satiety signals. Moreover, mRNA expression of all three CART-1, CART-2, and CART-3 was significantly reduced during fasting and significantly elevated with refeeding. Our findings indicate that CART-1, CART-2, and CART-3 might function as a satiety factor in the gibel carp.


Assuntos
Comportamento Alimentar/fisiologia , Carpa Dourada/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Filogenia , Isoformas de Proteínas
13.
Dis Markers ; 2019: 8282414, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31089399

RESUMO

Objective: The synaptic adhesion-like molecule (SALM) family is largely restricted to neural tissues and is involved in the regulation of neurite outgrowth and synapse formation. However, the expression of SALM3 in gastric cancer (GC) and its clinical significance remain unclear. The aim of the present study was to investigate the prognostic value of SALM3 in patients with GC. Patients and Methods: Expression of SALM3 was validated by tissue microarrays from 730 GC patients and statistically assessed for correlations with the clinical parameters and the prognosis of the patients. The transcriptional and survival data of SALM3 in GC patients were also mined through the Oncomine and Kaplan-Meier Plotter databases. Results: SALM3 is overexpressed in the tumor cells and fibroblasts of clinical GC tissues, and a high level of SALM3 was significantly associated with tumor invasive characteristics. Cox proportional hazards univariate and multivariate regression analyses revealed SALM3 expression in tumor cells or stroma as an independent prognostic factor in the overall survival rate of GC patients. Furthermore, the survival of GC patients with high SALM3 expression in both tumor cells and fibroblasts was significantly poorer than that of the other groups. Oncomine and Kaplan-Meier Plotter analyses further confirmed high levels of SALM3 expression in GC, and high levels of SALM3 expression were associated with shorter survival in patients. Conclusion: SALM3 may be a prognostic factor for GC and may potentially be a high-priority therapeutic target.


Assuntos
Biomarcadores Tumorais/genética , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neoplasias Gástricas/metabolismo , Biomarcadores Tumorais/metabolismo , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
14.
Nat Commun ; 10(1): 2350, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138815

RESUMO

Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions.


Assuntos
Aciltransferases/genética , Células Endoteliais/metabolismo , Neovascularização Fisiológica/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular/genética , Polaridade Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Endocitose/genética , Células Endoteliais/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Vasos Retinianos/citologia , Vasos Retinianos/crescimento & desenvolvimento , Quinases Ativadas por p21/metabolismo
15.
Molecules ; 24(9)2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31052478

RESUMO

The synaptic vesicle protein 2 (SV2) is involved in synaptic vesicle trafficking. The SV2A isoform is the most studied and its implication in epilepsy therapy led to the development of the first SV2A PET radiotracer [18F]UCB-H. The objective of this study was to evaluate in vivo, using microPET in rats, the specificity of [18F]UCB-H for SV2 isoform A in comparison with the other two isoforms (B and C) through a blocking assay. Twenty Sprague Dawley rats were pre-treated either with the vehicle, or with specific competitors against SV2A (levetiracetam), SV2B (UCB5203) and SV2C (UCB0949). The distribution volume (Vt, Logan plot, t* 15 min) was obtained with a population-based input function. The Vt analysis for the entire brain showed statistically significant differences between the levetiracetam group and the other groups (p < 0.001), but also between the vehicle and the SV2B group (p < 0.05). An in-depth Vt analysis conducted for eight relevant brain structures confirmed the statistically significant differences between the levetiracetam group and the other groups (p < 0.001) and highlighted the superior and the inferior colliculi along with the cortex as regions also displaying statistically significant differences between the vehicle and SV2B groups (p < 0.05). These results emphasize the in vivo specificity of [18F]UCB-H for SV2A against SV2B and SV2C, confirming that [18F]UCB-H is a suitable radiotracer for in vivo imaging of the SV2A proteins with PET.


Assuntos
Encéfalo/diagnóstico por imagem , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Piridinas/metabolismo , Pirrolidinonas/metabolismo , Animais , Encéfalo/metabolismo , Levetiracetam/administração & dosagem , Levetiracetam/farmacologia , Imagem por Ressonância Magnética , Masculino , Modelos Animais , Estrutura Molecular , Tomografia por Emissão de Pósitrons , Piridinas/química , Pirrolidinonas/química , Ratos , Ratos Sprague-Dawley , Sensibilidade e Especificidade
16.
Nat Commun ; 10(1): 2232, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110186

RESUMO

Sparse populations of neurons in the dentate gyrus (DG) of the hippocampus are causally implicated in the encoding of contextual fear memories. However, engram-specific molecular mechanisms underlying memory consolidation remain largely unknown. Here we perform unbiased RNA sequencing of DG engram neurons 24 h after contextual fear conditioning to identify transcriptome changes specific to memory consolidation. DG engram neurons exhibit a highly distinct pattern of gene expression, in which CREB-dependent transcription features prominently (P = 6.2 × 10-13), including Atf3 (P = 2.4 × 10-41), Penk (P = 1.3 × 10-15), and Kcnq3 (P = 3.1 × 10-12). Moreover, we validate the functional relevance of the RNAseq findings by establishing the causal requirement of intact CREB function specifically within the DG engram during memory consolidation, and identify a novel group of CREB target genes involved in the encoding of long-term memory.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/metabolismo , Giro Denteado/fisiologia , Consolidação da Memória/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Animais , Condicionamento (Psicologia)/fisiologia , Giro Denteado/citologia , Encefalinas/genética , Encefalinas/metabolismo , Medo/fisiologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/fisiologia , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Neurônios/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Análise de Sequência de RNA , Técnicas Estereotáxicas
17.
Nat Neurosci ; 22(6): 897-908, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086315

RESUMO

Developmental programs that generate the astonishing neuronal diversity of the nervous system are not completely understood and thus present a major challenge for clinical applications of guided cell differentiation strategies. Using direct neuronal programming of embryonic stem cells, we found that two main vertebrate proneural factors, Ascl1 and neurogenin 2 (Neurog2), induce different neuronal fates by binding to largely different sets of genomic sites. Their divergent binding patterns are not determined by the previous chromatin state, but are distinguished by enrichment of specific E-box sequences that reflect the binding preferences of the DNA-binding domains. The divergent Ascl1 and Neurog2 binding patterns result in distinct chromatin accessibility and enhancer activity profiles that differentially shape the binding of downstream transcription factors during neuronal differentiation. This study provides a mechanistic understanding of how transcription factors constrain terminal cell fates, and it delineates the importance of choosing the right proneural factor in neuronal reprogramming strategies.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromatina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neurônios/citologia , Animais , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias , Humanos , Neurônios/metabolismo
18.
Cell Physiol Biochem ; 52(6): 1361-1380, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31075188

RESUMO

BACKGROUND/AIMS: Human Dental Pulp Stem Cells (hDPSCs) are one of the most promising types of cells to regenerate nerve tissues. Standard DMEM+10% fetal bovine serum (FBS) culture medium allows a fast expansion of hDPSC as a surface-adherent cell monolayer. However, the use of FBS also compromises the clinical use of these protocols, and its longterm presence favors hDPSCs differentiation toward mesenchymal cell-derived lineages, at the expense of a reduced capability to generate neural cells. The objective of this work was to characterize the role of neurotrophin signaling on hDPSCs using a serum-free culture protocol, and to assess the neurogenic and gliogenic capacity of hDPSCs for future nerve tissue bioengineering and regeneration. METHODS: We compared the different expression of neurotrophin receptors by RT-PCR, Q-PCR, and IF of hDPSCs cultured with different growth media in the presence or absence of serum. Moreover, we assessed the response of hDPSCs to stimulation of neurotransmitter receptors by live cell calcium imaging under these different media. Finally, we compared the osteogenic potential of hDPSCs by Alizarin red staining, and the differentiation to gliogenic/neurogenic fates by immunostaining for Schwann lineage and neuronal lineage markers. We tested a commercial serum-free medium designed for the growth of mesenchymal stem cells: StemPro MSCTM (STP). RESULTS: hDPSCs cultured in STP generated small non-adherent floating dentospheres that showed very low proliferation rates, in contrast to standard FBS-containing medium. We found that hDPSCs grown in STP conditions overexpressed neurotrophin receptor genes NTRK2 (TrkB) and NTRK3 (TrkC). Interestingly, the stimulation of these receptors by adding their respective ligands BDNF and NT-3 to STP medium enhanced the neural crest (NC) progenitor features of cultured hDPSCs. We observed a 10 to 100-fold increase of migratory NC cell markers HNK1 and P75NTR, and a significant overexpression of pluripotency core factors SOX2, OCT4 and NANOG. Moreover, hDPSCs cultured in BDNF/NT-3 supplemented STP showed a largely increased potential to differentiate towards neuronal and Schwann glial lineage cells, assessed by positive immunostaining for DCX, NeuN and S100ß, p75NTR markers, respectively. CONCLUSION: Our results demonstrate that the use of BDNF and NT-3 combined with STP induced the partial reprogramming of ectomesenchymal hDPSCs to generate early NC progenitor cells, which are far more competent for neuronal and glial differentiation than hDPSCs grown in the presence of FBS.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Reprogramação Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Fatores de Crescimento Neural/farmacologia , Adolescente , Adulto , Antígenos CD57/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Polpa Dentária/citologia , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Crista Neural/citologia , Neurogênese/efeitos dos fármacos , Receptor trkA/genética , Receptor trkA/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Adulto Jovem
19.
DNA Cell Biol ; 38(7): 700-707, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31090452

RESUMO

Substantial research has revealed that peroxisome proliferator-activated receptor-gamma (PPARG) plays a critical role in glucose homeostasis and lipid metabolism, and recent studies have shown different effects in the progression of different tumors. However, the role of PPARG and its target gene in clear cell renal cell carcinoma (ccRCC) are incompletely understood. Clinical data revealed abnormal glucolipid metabolism in primary ccRCC samples. In addition, transcriptional profiling indicated that PPARG expression was positively correlated, whereas Six2 expression was negatively correlated with the overall survival of ccRCC patients. Staining showed that PPARG was mainly expressed in tumor cell cytoplasm, and Six2 was localized to the nuclei. In a ccRCC cell line, PPARG activation promoted cell apoptosis, inhibited cell migration and proliferation, and reduced Six2 expression. Mechanistically, overexpressing Six2 downregulated E-cadherin expression and cell apoptosis, but PPARG activation reversed those effects. Taken together, PPARG promotes apoptosis and suppresses the migration and proliferation of ccRCC cells by inhibiting Six2. These findings reveal that the PPARG/Six2 axis acts as a central pathobiological mediator of ccRCC formation and as a potential therapeutic target for the treatment of patients with ccRCC.


Assuntos
Carcinoma de Células Renais/metabolismo , Proteínas de Homeodomínio/genética , Neoplasias Renais/metabolismo , Proteínas do Tecido Nervoso/genética , PPAR gama/genética , Apoptose , Caderinas/genética , Caderinas/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Feminino , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , PPAR gama/metabolismo
20.
J Neuroinflammation ; 16(1): 67, 2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30927920

RESUMO

BACKGROUND: Patients diagnosed with chronic fatigue syndrome (CFS) or fibromyalgia experience chronic pain. Concomitantly, the rat model of CFS exhibits microglial activation in the lumbar spinal cord and pain behavior without peripheral tissue damage and/or inflammation. The present study addressed the mechanism underlying the association between pain and chronic stress using this rat model. METHODS: Chronic or continuous stress-loading (CS) model rats, housed in a cage with a thin level of water (1.5 cm in depth), were used. The von Frey test and pressure pain test were employed to measure pain behavior. The neuronal and microglial activations were immunohistochemically demonstrated with antibodies against ATF3 and Iba1. Electromyography was used to evaluate muscle activity. RESULTS: The expression of ATF3, a marker of neuronal hyperactivity or injury, was first observed in the lumbar dorsal root ganglion (DRG) neurons 2 days after CS initiation. More than 50% of ATF3-positive neurons simultaneously expressed the proprioceptor markers TrkC or VGluT1, whereas the co-expression rates for TrkA, TrkB, IB4, and CGRP were lower than 20%. Retrograde labeling using fluorogold showed that ATF3-positive proprioceptive DRG neurons mainly projected to the soleus. Substantial microglial accumulation was observed in the medial part of the dorsal horn on the fifth CS day. Microglial accumulation was observed around a subset of motor neurons in the dorsal part of the ventral horn on the sixth CS day. The motor neurons surrounded by microglia were ATF3-positive and mainly projected to the soleus. Electromyographic activity in the soleus was two to three times higher in the CS group than in the control group. These results suggest that chronic proprioceptor activation induces the sequential activation of neurons along the spinal reflex arc, and the neuronal activation further activates microglia along the arc. Proprioceptor suppression by ankle joint immobilization significantly suppressed the accumulation of microglia in the spinal cord, as well as the pain behavior. CONCLUSION: Our results indicate that proprioceptor-induced microglial activation may be a key player in the initiation and maintenance of abnormal pain in patients with CFS.


Assuntos
Citocinas/metabolismo , Síndrome de Fadiga Crônica/complicações , Microglia/patologia , Dor/etiologia , Dor/patologia , Distúrbios Somatossensoriais/etiologia , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Gânglios Espinais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Medição da Dor , Ratos , Ratos Sprague-Dawley , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Distúrbios Somatossensoriais/patologia , Estilbamidinas/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo
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