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1.
Cell ; 185(21): 3931-3949.e26, 2022 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-36240740

RESUMO

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.


Assuntos
Movimento Celular , Glipicanas/química , Receptores de Netrina/química , Animais , Glipicanas/metabolismo , Humanos , Camundongos , Proteínas Mutantes , Receptores de Netrina/metabolismo , Receptores de Superfície Celular/metabolismo , Anticorpos de Domínio Único , Trombospondinas
2.
Proc Natl Acad Sci U S A ; 117(39): 24503-24513, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32929029

RESUMO

The Hippo (MST1/2) pathway plays a critical role in restricting tissue growth in adults and modulating cell proliferation, differentiation, and migration in developing organs. Netrin1, a secreted laminin-related protein, is essential for nervous system development. However, the mechanisms underlying MST1 regulation by the extrinsic signals remain unclear. Here, we demonstrate that Netrin1 reduction in Parkinson's disease (PD) activates MST1, which selectively binds and phosphorylates netrin receptor UNC5B on T428 residue, promoting its apoptotic activation and dopaminergic neuronal loss. Netrin1 deprivation stimulates MST1 activation and interaction with UNC5B, diminishing YAP levels and escalating cell deaths. Knockout of UNC5B abolishes netrin depletion-induced dopaminergic loss, whereas blockade of MST1 phosphorylating UNC5B suppresses neuronal apoptosis. Remarkably, Netrin1 is reduced in PD patient brains, associated with MST1 activation and UNC5B T428 phosphorylation, which is accompanied by YAP reduction and apoptotic activation. Hence, Netrin1 regulates Hippo (MST1) pathway in dopaminergic neuronal loss in PD via UNC5B receptor.


Assuntos
Apoptose , Neurônios Dopaminérgicos/citologia , Receptores de Netrina/metabolismo , Netrina-1/metabolismo , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Proliferação de Células , Neurônios Dopaminérgicos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Receptores de Netrina/química , Receptores de Netrina/genética , Netrina-1/genética , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética
3.
Cell Commun Signal ; 20(1): 122, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35974411

RESUMO

BACKGROUND: B-cell acute lymphoblastic leukemia (B-ALL) comprises over 85% of all acute lymphoblastic leukemia (ALL) cases and is the most common childhood malignancy. Although the 5 year overall survival of patients with B-ALL exceeds 90%, patients with relapsed or refractory B-ALL may suffer from poor prognosis and adverse events. The axon guidance factor netrin-1 has been reported to be involved in the tumorigenesis of many types of cancers. However, the impact of netrin-1 on B-ALL remains unknown. METHODS: The expression level of netrin-1 in peripheral blood samples of children with B-ALL and children without neoplasia was measured by enzyme-linked immunosorbent assay (ELISA) kits. Then, CCK-8 cell proliferation assays and flow cytometric analysis were performed to detect the viability and apoptosis of B-ALL cells (Reh and Sup B15) treated with exogenous recombinant netrin-1 at concentrations of 0, 25, 50, and 100 ng/ml. Furthermore, co-immunoprecipitation(co-IP) was performed to detect the receptor of netrin-1. UNC5B expression interference was induced in B-ALL cells with recombinant lentivirus, and then CCK-8 assays, flow cytometry assays and western blotting assays were performed to verify that netrin-1 might act on B-ALL cells via the receptor Unc5b. Finally, western blotting and kinase inhibitor treatment were applied to detect the downstream signaling pathway. RESULTS: Netrin-1 expression was increased in B-ALL, and netrin-1 expression was upregulated in patients with high- and intermediate-risk stratification group of patients. Then, we found that netrin-1 induced an anti-apoptotic effect in B-ALL cells, implying that netrin-1 plays an oncogenic role in B-ALL. co-IP results showed that netrin-1 interacted with the receptor Unc5b in B-ALL cells. Interference with UNC5B was performed in B-ALL cells and abolished the antiapoptotic effects of netrin-1. Further western blotting was applied to detect the phosphorylation levels of key molecules in common signaling transduction pathways in B-ALL cells treated with recombinant netrin-1, and the FAK-MAPK signaling pathway was found to be activated. The anti-apoptotic effect of netrin-1 and FAK-MAPK phosphorylation was abrogated by UNC5B interference. FAK inhibitor treatment and ERK inhibitor treatment were applied and verified that the FAK-MAPK pathway may be downstream of Unc5b. CONCLUSION: Taken together, our findings suggested that netrin-1 induced the anti-apoptotic effect of B-ALL cells through activation of the FAK-MAPK signaling pathway by binding to the receptor Unc5b. Video Abstract.


Assuntos
Receptores de Netrina , Netrina-1 , Leucemia-Linfoma Linfoblástico de Células Precursoras , Criança , Humanos , Sistema de Sinalização das MAP Quinases , Receptores de Netrina/metabolismo , Netrina-1/metabolismo , Netrina-1/farmacologia , Receptores de Superfície Celular/metabolismo , Sincalida , Proteínas Supressoras de Tumor/metabolismo
4.
J Cell Mol Med ; 25(4): 2121-2135, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33345442

RESUMO

The intracellular domain of UNC5B contains both death domain and caspase-3 cleavage site, and is regarded as a functional domain that mediates apoptosis. However, in our previous studies, we found that the death domain of UNC5B in bladder cancer cells could not be activated to promote apoptosis. In this study, different UNC5B truncates (residue 399-945, residue 412-945) were created to explore whether the caspase-3 cleavage site (site 412), as another potential functional domain of its intracellular portion, could be activated to induce apoptosis in bladder cancer cells. Using mass spectrometry, we acquired a comprehensive and detailed identification of differentially expressed proteins by overexpressing UNC5B and its truncates. Protein-protein-interaction (PPI) network analysis was also applied to investigate the aggregation of related proteins and predict the functional changes. EDU assay, apoptosis, xenograft tumour implantation, migration, invasion and tumour metastasis were performed to comprehensively identify the effects of UNC5B truncates on bladder cancer cells. We demonstrate that the intracellular domain of UNC5B promotes cell proliferation in vitro and tumour formation in vivo, by binding to a large number of ribosomal proteins. The overexpression of intracellular domain also facilitates cells to migrate, invade and metastasize by interacting with fibronectin, beta-catenin and vimentin. In addition, we reveal that overexpressing the intracellular domain of UNC5B cannot bind or activate cleaved caspase-3 to trigger apoptosis in bladder cancer cells.


Assuntos
Receptores de Netrina/metabolismo , Domínios Proteicos , Neoplasias da Bexiga Urinária/metabolismo , Animais , Apoptose , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Biologia Computacional/métodos , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Receptores de Netrina/química , Receptores de Netrina/genética , Domínios Proteicos/genética , Proteômica/métodos , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo
5.
J Gene Med ; 23(12): e3382, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34350661

RESUMO

BACKGROUND: Long non-coding RNAs (lncRNAs) are significant regulatory factors for the initiation and development of numerous malignant tumors, including cervical cancer (CC). The expression of lncRNA unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1, also known as UASR1) is up-regulated in tissues of cervical squamous cell carcinoma and endocervical adenocarcinoma compared to in normal tissues based on the GEPIA database. In the present study, we explored the functions of UNC5B-AS1 and its underlying mechanism with respect to CC development. METHODS: A real-time quantitative polymerase chain reaction was applied for the detection of UNC5B-AS1 expression in CC cells. Cell counting kit-8, colony formation and transwell assays, as well as western blot and flow cytometry analyses, were employed to detect the biological effects of UNC5B-AS1 knockdown on malignant phenotypes of CC cells in vitro. In addition, the combination between microRNA-4455 (miR-4455) and UNC5B-AS1 or R-spondin 4 (RSPO4) was explored by RNA immunoprecipitation, luciferase reporter and RNA pulldown assays. A tumor xenograft nude mice model was established to explore the effect of UNC5B-AS1 depletion or miR-4455 overexpression on tumor growth. RESULTS: UNC5B-AS1 is up-regulated in CC tissues and cells. The knockdown of UNC5B-AS1 inhibits CC cell proliferation, migration and invasion and promotes CC cell apoptosis. Mechanistically, UNC5B-AS1 binds with miR-4455 to up-regulate RSPO4 expression. RSPO4 is targeted by miR-4455 and its expression is negatively regulated by miR-4455 expression. In vivo assays revealed that UNC5B-AS1 depletion or miR-4455 overexpression inhibits tumor growth by regulating RSPO4 expression. CONCLUSIONS: Inhibition of UNC5B-AS1/miR-4455/RSPO4 reduces CC growth both in vitro and in vivo, furnishing new insights into molecular studies on UNC5B-AS1 with respect to CC development.


Assuntos
MicroRNAs , RNA Longo não Codificante , Neoplasias do Colo do Útero , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Receptores de Netrina/genética , Receptores de Netrina/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
6.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32404521

RESUMO

UNC5B is a dependence receptor that promotes survival in the presence of its ligand, netrin-1, while inducing cell death in its absence. The receptor has an important role in the development of the nervous and vascular systems. It is also involved in the normal turnover of intestinal epithelium. Netrin-1 and UNC5B are deregulated in multiple cancers, including colorectal, neuroblastoma, and breast tumors. However, the detailed mechanism of UNC5B function is not fully understood. We have utilized the murine polyomavirus small T antigen (PyST) as a tool to study UNC5B-mediated apoptosis. PyST is known to induce mitotic arrest followed by extensive cell death in mammalian cells. Our results show that the expression of PyST increases mRNA levels of UNC5B by approximately 3-fold in osteosarcoma cells (U2OS) and also stabilizes UNC5B at the posttranslational level. Furthermore, UNC5B is upregulated predominantly in those cells that undergo mitotic arrest upon PyST expression. Interestingly, although its expression was previously reported to be regulated by p53, our data show that the increase in UNC5B levels by PyST is p53 independent. The posttranslational stabilization of UNC5B by PyST is regulated by the interaction of PyST with PP2A. We also show that netrin-1 expression, which is known to inhibit UNC5B apoptotic activity, promotes survival of PyST-expressing cells. Our results thus suggest an important role of UNC5B in small-T antigen-induced mitotic catastrophe that also requires PP2A.IMPORTANCE UNC5B, PP2A, and netrin-1 are deregulated in a variety of cancers. UNC5B and PP2A are regarded as tumor suppressors, as they promote apoptosis and are deleted or mutated in many cancers. In contrast, netrin-1 promotes survival by inhibiting dependence receptors, including UNC5B, and is upregulated in many cancers. Here, we show that UNC5B-mediated apoptosis can occur independently of p53 but in a PP2A-dependent manner. A substantial percentage of cancers arise due to p53 mutations and are insensitive to chemotherapeutic treatments that activate p53. Unexpectedly, treatment of cancers having functional p53 with many conventional drugs leads to the upregulation of netrin-1 through activated p53, which is counterintuitive. Therefore, understanding the p53-independent mechanisms of the netrin-UNC5B axis, such as those involving PP2A, assumes greater clinical significance. Anticancer strategies utilizing anti-netrin-1 antibody treatment are already in clinical trials.


Assuntos
Antígenos Virais de Tumores/metabolismo , Apoptose , Receptores de Netrina/metabolismo , Polyomavirus/metabolismo , Proteína Fosfatase 2/metabolismo , Células A549 , Animais , Antígenos Virais de Tumores/genética , Células HeLa , Humanos , Camundongos , Receptores de Netrina/genética , Polyomavirus/genética , Proteína Fosfatase 2/genética
7.
J Biol Chem ; 294(19): 7669-7681, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30910812

RESUMO

Although cannabinoid receptor 1 (CB1) antagonists have been shown to attenuate diet-induced obesity (DIO) and associated inflammation, the precise molecular mechanisms involved are not clear. In the current study, we investigated the role of microRNA (miR) in the regulation of adipose tissue macrophage (ATM) phenotype following treatment of DIO mice with the CB1 antagonist SR141716A. DIO mice were fed high-fat diet (HFD) for 12 weeks and then treated daily with SR141716A (10 mg/kg) for 4 weeks while continuing HFD. Treated mice experienced weight loss, persistent reduction in fat mass, improvements in metabolic profile, and decreased adipose inflammation. CB1 blockade resulted in down-regulation of several miRs in ATMs, including the miR-466 family and miR-762. Reduced expression of the miR-466 family led to induction of anti-inflammatory M2 transcription factors KLF4 and STAT6, whereas down-regulation of miR-762 promoted induction of AGAP-2, a negative regulator of the neuroimmune retention cues, Netrin-1 and its coreceptor UNC5B. Furthermore, treatment of primary macrophages with SR141716A up-regulated KLF4 and STAT6, reduced secretion of Netrin-1, and increased migration toward the lymph node chemoattractant CCL19. These studies demonstrate for the first time that CB1 receptor blockade attenuates DIO-associated inflammation through alterations in ATM miR expression that promote M2 ATM polarization and macrophage egress from adipose tissue. The current study also identifies additional novel therapeutic targets for diet-induced obesity and metabolic disorder.


Assuntos
Tecido Adiposo/metabolismo , Quimiotaxia/efeitos dos fármacos , Gorduras na Dieta/efeitos adversos , Macrófagos/metabolismo , MicroRNAs/metabolismo , Obesidade/metabolismo , Receptor CB1 de Canabinoide/antagonistas & inibidores , Rimonabanto/farmacologia , Tecido Adiposo/patologia , Animais , Gorduras na Dieta/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/biossíntese , Macrófagos/patologia , Masculino , Camundongos , Obesidade/induzido quimicamente , Obesidade/tratamento farmacológico , Receptor CB1 de Canabinoide/metabolismo , Fator de Transcrição STAT6/biossíntese
8.
Development ; 144(13): 2392-2401, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28576770

RESUMO

The placental labyrinth is the interface for gas and nutrient exchange between the embryo and the mother; hence its proper development is essential for embryogenesis. However, the molecular mechanism underlying development of the placental labyrinth, particularly in terms of its endothelial organization, is not well understood. Here, we determined that fibronectin leucine-rich transmembrane protein 2 (FLRT2), a repulsive ligand of the UNC5 receptor family for neurons, is unexpectedly expressed in endothelial cells specifically in the placental labyrinth. Mice lacking FLRT2 in endothelial cells exhibited embryonic lethality at mid-gestation, with systemic congestion and hypoxia. Although they lacked apparent deformities in the embryonic vasculature and heart, the placental labyrinths of these embryos exhibited aberrant alignment of endothelial cells, which disturbed the feto-maternal circulation. Interestingly, this vascular deformity was related to endothelial repulsion through binding to the UNC5B receptor. Our results suggest that the proper organization of the placental labyrinth depends on coordinated inter-endothelial repulsion, which prevents uncontrolled layering of the endothelium.


Assuntos
Glicoproteínas de Membrana/metabolismo , Organogênese , Placenta/embriologia , Placenta/metabolismo , Transdução de Sinais , Animais , Sobrevivência Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Feminino , Deleção de Genes , Hipóxia/patologia , Glicoproteínas de Membrana/deficiência , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Receptores de Netrina , Placenta/irrigação sanguínea , Placenta/citologia , Gravidez , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/metabolismo
9.
Cell Biol Int ; 44(4): 1028-1036, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31903696

RESUMO

The role of long non-coding RNAs (lncRNAs) in tumorigenesis and development of ovarian cancer (OC) has caught the attention of scientists. UNC5B antisense RNA 1 (UNC5B-AS1) is a newly identified carcinogenic lncRNA in thyroid papillary carcinoma, but its role in OC remains unclear. This study is proposed to investigate the function and mechanism of UNC5B-AS1 in OC. UNC5B-AS1 expression in OC samples was obtained from gene expression profiling interactive analysis (GEPIA) based on The Cancer Genome Atlas data. Gene expressions were detected by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. Biological functions of UNC5B-AS1 were assessed by cell counting kit-8, colony formation, and caspase-3 analysis. GEPIA revealed the UNC5B-AS1 upregulation in OC samples. RT-qPCR assay confirmed the upregulation of UNC5B-AS1 in OC cells. Functionally, depletion of UCN5B-AS1 hindered proliferation and prompted apoptosis in OC cells. Mechanistically, we found that UNC5B-AS1 interacted with zeste 2 polycomb repressive complex 2 subunit (EZH2) to trigger trimethylation of histone H3 at lysine 27 (H3K27me3) on N-myc downstream regulated gene-2 (NDRG2) promoter and epigenetically repressed NDRG2. Rescue assay indicated the participation of NDRG2 in the regulation of UNC5B-AS1 on OC progression. Together, we first illustrated that UNC5B-AS1 promoted OC progression by regulating the H3K27me on NDRG2 via EZH2, indicating UNC5B-AS1 as a potential molecular target for OC treatment.


Assuntos
Receptores de Netrina/genética , Neoplasias Ovarianas/patologia , RNA Antissenso/genética , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Proteínas Supressoras de Tumor/metabolismo
10.
Int J Mol Sci ; 21(16)2020 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-32784443

RESUMO

Multinucleation is a hallmark of osteoclast maturation. The unique and dynamic multinucleation process not only increases cell size but causes functional alterations through reconstruction of the cytoskeleton, creating the actin ring and ruffled border that enable bone resorption. Our understanding of the molecular mechanisms underlying osteoclast multinucleation has advanced considerably in this century, especially since the identification of DC-STAMP and OC-STAMP as "master fusogens". Regarding the molecules and pathways surrounding these STAMPs, however, only limited progress has been made due to the absence of their ligands. Various molecules and mechanisms other than the STAMPs are involved in osteoclast multinucleation. In addition, several preclinical studies have explored chemicals that may be able to target osteoclast multinucleation, which could enable us to control pathogenic bone metabolism more precisely. In this review, we will focus on recent discoveries regarding the STAMPs and other molecules involved in osteoclast multinucleation.


Assuntos
Núcleo Celular/metabolismo , Osteoclastos/citologia , Animais , Humanos , Modelos Biológicos , Osteoclastos/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais
11.
Int J Mol Sci ; 20(6)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897795

RESUMO

Gestational diabetes mellitus (GDM) is a common metabolic disorder, defined by high blood glucose levels during pregnancy, which affects foetal and post-natal development. However, the cellular and molecular mechanisms of this detrimental condition are still poorly understood. A dysregulation in circulating angiogenic trophic factors, due to a dysfunction of the feto-placental unit, has been proposed to underlie GDM. But even the detailed study of canonical pro-angiogenic factors like vascular endothelial growth factor (VEGF) or basic Fibroblast Growth Factor (bFGF) has not been able to fully explain this detrimental condition during pregnancy. Netrins are non-canonical angiogenic ligands produced by the stroma have shown to be important in placental angiogenesis. In order to address the potential role of Netrin signalling in GDM, we tested the effect of Netrin-1, the most investigated member of the family, produced by Wharton's Jelly Mesenchymal Stem Cells (WJ-MSC), on Human Umbilical Vein Endothelial Cells (HUVEC) angiogenesis. WJ-MSC and HUVEC primary cell cultures from either healthy or GDM pregnancies were exposed to physiological (5 mM) or high (25 mM) d-glucose. Our results reveal that Netrin-1 is secreted by WJ-MSC from healthy and GDM and both expression and secretion of the ligand do not change with distinct experimental glucose conditions. Noteworthy, the expression of its anti-angiogenic receptor UNC5b is reduced in GDM HUVEC compared with its expression in healthy HUVEC, accounting for an increased Netrin-1 signalling in these cells. Consistently, in healthy HUVEC, UNC5b overexpression induces cell retraction of the sprouting phenotype.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Netrina-1/metabolismo , Receptores de Superfície Celular/metabolismo , Diabetes Gestacional/genética , Diabetes Gestacional/metabolismo , Feminino , Humanos , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Receptores de Netrina , Netrina-1/genética , Gravidez , Receptores de Superfície Celular/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
J Cell Biochem ; 119(10): 8304-8316, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29893424

RESUMO

To investigate the biological functions and regulatory mechanism of lncRNA TNRC6C-AS1 in thyroid cancer (TC). TNRC6C-AS1, miR-129-5p, and UNC5B expression levels were investigated by qRT-PCR and Western blot. CCK-8 assay was conducted to determine cell proliferation, while transwell assay was for inspection of cell migration and invasion. Through bioinformatic analysis, the interactions among TNRC6C-AS1, miR-129-5p, and UNC5B were predicted. Dual luciferase reporter gene assay and RNA pull-down assay confirmed the predicted target relationships. Tumor xenograft assay was applied to inspect the effect of TNRC6C-AS1 downregulation on TC development in vivo. TNRC6C-AS1 and UNC5B were overexpressed, while miR-129-5p was underexpressed in TC tissues and cells. TNRC6C-AS1/UNC5B downregulation and miR-129-5p overexpression could suppress proliferation, migration, and invasion of TC cells as well as inhibit tumorigenesis in vivo. MiR-129-5p targeted TNRC6C-AS1 and UNC5B in TC cells; and UNC5B expression was downregulated by knocking down TNRC6C-AS1, which competitively bound with miR-129-5p. Downregulation of TNRC6C-AS1 restrained TC development by knocking down UNC5B through upregulating the expression of miR-129-5p.


Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Receptores de Superfície Celular/genética , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Adulto , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Metástase Linfática , Masculino , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica , Receptores de Netrina , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Câncer Papilífero da Tireoide/metabolismo , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Biochem Biophys Res Commun ; 505(3): 637-643, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30286954

RESUMO

The formation of macrophage foam cells by ingesting ox-LDL and focal retention in the subendothelial space are the hallmarks of the early atherosclerotic lesion. The C-C chemokine receptor type 7 (CCR7) is positively correlated with the macrophage migration. But the mechanism of CCR7 regulation is not fully clearness. In the present study, we demonstrates that expression in UNC5b and netrin-1 was enhanced in respond to ox-LDL in Raw264.7 macrophage and associated with decreasing cell migration. Interestingly, it was found that ox-LDL significantly downregulate CCR7 gene expression. The expression of CCR7 in mRNA and protein levels were decreased in ox-LDL treated Raw264.7 macrophage when we over expression of UNC5b with pcDNA3.1-UNC5b plasmid. We got the inverse results after silence UNC5b gene with siUNC5b. Meanwhile, the data show that in ox-LDL inducement, UNC5b down-regulated CCR7, and then inhibited macrophage migration. This novel phenomenon is of a crucial highlights to understand deeply the pathogenesis of atherosclerosis. The molecular mechanism of CCR7 regulation deserves intensive study.


Assuntos
Movimento Celular/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Receptores de Netrina/metabolismo , Receptores CCR7/metabolismo , Animais , Movimento Celular/genética , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Receptores de Netrina/genética , Netrina-1/genética , Netrina-1/metabolismo , Células RAW 264.7 , Interferência de RNA , Receptores CCR7/genética
14.
BMC Cancer ; 16(1): 892, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27846823

RESUMO

BACKGROUND: The netrin-1 receptor UNC5B plays vital roles in angiogenesis, inflammation, embryonic development and carcinogenesis. However, the functional significance of UNC5B overexpression in bladder cancer remains unclear. In this study, we investigated the role of UNC5B in bladder cancer in vitro and in vivo. METHODS: Stable transfection of the human bladder cancer cell line 5637 with UNC5B (5637-U) was confirmed by real-time RT-PCR, western blot and immunofluorescence assays. UNC5B expression in 5637 and 5637-U cells and mice tumor specimens derived from these cell lines was analyzed by immunohistochemistryand western blotting. Changes in the levels of cell cycle proteins were evaluated by western blotting. Flow cytometry, CCK-8 and scratch tests were used to examine cell cycle distribution, proliferation and migration, respectively. RESULTS: UNC5B overexpression in 5637 cells inhibited cell multiplication and migration and induced cell cycle arrest at the G2/M phase, meanwhile exhibited changes in the expression of cell cycle-associated proteins, showing that UNC5B may inhibit metastatic behaviors in bladder cancer cells. In addition, tumors generated from 5637-U cells were smaller than tumors generated from control 5637 cells. CONCLUSIONS: Our findings suggest that UNC5B is a potential anti-neoplastic target in bladder cancer progression.


Assuntos
Expressão Gênica , Receptores de Superfície Celular/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Modelos Animais de Doenças , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Receptores de Netrina , Transporte Proteico , Receptores de Superfície Celular/metabolismo , Carga Tumoral
15.
Biochem Biophys Res Commun ; 464(1): 263-8, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26116534

RESUMO

Schwann cells (SCs) play an essentially supportive role in the regeneration of injured peripheral nerve system (PNS). As Netrin-1 is crucial for the normal development of nervous system (NS) and can direct the process of damaged PNS regeneration, our study was designed to determine the role of Netrin-1 in RSC96 Schwann cells (an immortalized rat Schwann cell line) proliferation and migration. Our studies demonstrated that Netrin-1 had no effect on RSC96 cells proliferation, while significantly promoted RSC96 cells migration. The Netrin-1-induced RSC96 cells migration was significantly attenuated by inhibition of p38 and PI3K through pretreatment with SB203580 and LY294002 respectively, but not inhibition of MEK1/2 and JNK by U0126-EtOH and SP600125 individually. Treatment with Netrin-1 enhanced the phosphorylation of p38 and Akt. QRT-PCR indicated that Netrin-1 and only its receptors Unc5a, Unc5b and Neogenin were expressed in RSC96 cells, among which Unc5b expressed the most. And UNC5B protein was significantly increased after stimulated by Netrin-1. In conclusion, we show here that Netrin-1-enhanced SCs migration is mediated by activating p38 MAPK and PI3K-Akt signal cascades via receptor UNC5B, which suggests that Netrin-1 could serve as a new therapeutic strategy and has potential application value for PNS regeneration.


Assuntos
Fatores de Crescimento Neural/farmacologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Superfície Celular/genética , Células de Schwann/efeitos dos fármacos , Proteínas Supressoras de Tumor/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Cromonas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica , Imidazóis/farmacologia , Camundongos , Morfolinas/farmacologia , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Netrina-1 , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/farmacologia , Ratos , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Células de Schwann/citologia , Células de Schwann/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
J Cell Mol Med ; 18(7): 1290-9, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24720832

RESUMO

The netrin-1 administration or overexpression is known to protect colon from acute colitis. However, the receptor that mediates netrin-1 protective activities in the colon during colitis remains unknown. We tested the hypothesis that UNC5B receptor is a critical mediator of protective function of netrin-1 in dextran sodium sulfate (DSS)-induced colitis using mice with partial deletion of UNC5B receptor. DSS colitis was performed in mice with partial genetic UNC5B deficiency (UNC5B(+/-) mice) or wild-type mice to examine the role of endogenous UNC5B. These studies were supported by in vitro models of DSS-induced apoptosis in human colon epithelial cells. WT mice developed colitis in response to DSS feeding as indicated by reduction in bw, reduction in colon length and increase in colon weight. These changes were exacerbated in heterozygous UNC5B knockout mice treated with DSS. Periodic Acid-Schiff stained section shows damages in colon epithelium and mononuclear cell infiltration in WT mice, which was further increased in UNC5B heterozygous knockout mice. This was associated with large increase in inflammatory mediators such as cytokine and chemokine expression and extensive apoptosis of epithelial cells in heterozygous knockout mice as compared to WT mice. Overexpression of UNC5B human colon epithelial cells suppressed DSS-induced apoptosis and caspase-3 activity. Moreover, DSS induced large amount of netrin-1 and shRNA mediated knockdown of netrin-1 induction exacerbated DSS-induced epithelial cell apoptosis. Our results suggest that UNC5B is a critical mediator of cell survival in response to stress in colon.


Assuntos
Colite/genética , Colite/patologia , Sulfato de Dextrana/toxicidade , Células Epiteliais/patologia , Receptores de Superfície Celular/fisiologia , Animais , Western Blotting , Colite/induzido quimicamente , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Citometria de Fluxo , Humanos , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Receptores de Netrina , Netrina-1 , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
17.
Sci Rep ; 14(1): 13603, 2024 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-38866944

RESUMO

Notch signaling guides vascular development and function by regulating diverse endothelial cell behaviors, including migration, proliferation, vascular density, endothelial junctions, and polarization in response to flow. Notch proteins form transcriptional activation complexes that regulate endothelial gene expression, but few of the downstream effectors that enable these phenotypic changes have been characterized in endothelial cells, limiting our understanding of vascular Notch activities. Using an unbiased screen of translated mRNA rapidly regulated by Notch signaling, we identified novel in vivo targets of Notch signaling in neonatal mouse brain endothelium, including UNC5B, a member of the netrin family of angiogenic-regulatory receptors. Endothelial Notch signaling rapidly upregulates UNC5B in multiple endothelial cell types. Loss or gain of UNC5B recapitulated specific Notch-regulated phenotypes. UNC5B expression inhibited endothelial migration and proliferation and was required for stabilization of endothelial junctions in response to shear stress. Loss of UNC5B partially or wholly blocked the ability of Notch activation to regulate these endothelial cell behaviors. In the developing mouse retina, endothelial-specific loss of UNC5B led to excessive vascularization, including increased vascular outgrowth, density, and branchpoint count. These data indicate that Notch signaling upregulates UNC5B as an effector protein to control specific endothelial cell behaviors and inhibit angiogenic growth.


Assuntos
Movimento Celular , Proliferação de Células , Células Endoteliais , Receptores de Netrina , Receptores Notch , Retina , Transdução de Sinais , Animais , Receptores de Netrina/metabolismo , Receptores Notch/metabolismo , Camundongos , Células Endoteliais/metabolismo , Retina/metabolismo , Humanos , Vasos Retinianos/metabolismo , Neovascularização Fisiológica
18.
Cell Rep ; 43(3): 113837, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38402584

RESUMO

Communication between adjacent endothelial cells is important for the homeostasis of blood vessels. We show that quiescent endothelial cells use Jagged1 to instruct neighboring endothelial cells to assume a quiescent phenotype and secure the endothelial barrier. This phenotype enforcement by neighboring cells is operated by R-Ras through activation of Akt3, which results in upregulation of a Notch ligand Jagged1 and consequential upregulation of Notch target genes, such as UNC5B, and VE-cadherin accumulation in the neighboring cells. These signaling events lead to the stable interaction between neighboring endothelial cells to continue to fortify juxtacrine signaling via Jagged1-Notch. This mode of intercellular signaling provides a positive feedback regulation of endothelial cell-cell interactions and cellular quiescence required for the stabilization of the endothelium.


Assuntos
Células Endoteliais , Proteínas de Membrana , Proteínas Serrate-Jagged , Células Endoteliais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação ao Cálcio/genética , Peptídeos e Proteínas de Sinalização Intercelular , Receptores Notch/metabolismo , Proteína Jagged-1/genética
19.
BMC Med Genomics ; 17(1): 83, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38594690

RESUMO

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary CNS tumor, characterized by high mortality and heterogeneity. However, the related lncRNA signatures and their target microRNA (miRNA) for GBM are still mostly unknown. Therefore, it is critical that we discover lncRNA markers in GBM and their biological activities. MATERIALS AND METHODS: GBM-related RNA-seq data were obtained from the Cancer Genome Atlas (TCGA) database. The "edger" R package was used for differently expressed lncRNAs (DELs) identification. Then, we forecasted prospective miRNAs that might bind to lncRNAs by Cytoscape software. Survival analysis of those miRNAs was examined by the starBase database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the miRNAs' target genes was conducted by the Gene Set Enrichment Analysis (GSEA) database and R software. Moreover, the proliferative ability of unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1) cells was evaluated by Cell Counting Kit-8 (CCK-8) analysis. Mechanistically, the regulatory interaction between UNC5B-AS1 and miRNA in GBM biological processes was studied using CCK-8 analysis. RESULTS: Our results indicated that overexpression of UNC5B-AS1 has been shown to suppress GBM cell growth. Mechanistically, miR-24-3p in GBM was able to alleviate the anti-oncogenic effects of UNC5B-AS1 on cell proliferation. CONCLUSION: The discovery of the novel UNC5B-AS1-miR-24-3p network suggests possible lncRNA and miRNA roles in the development of GBM, which may have significant ramifications for the analysis of clinical prognosis and the development of GBM medications.


Assuntos
Glioblastoma , MicroRNAs , RNA Longo não Codificante , Humanos , Glioblastoma/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Estudos Prospectivos , Linhagem Celular Tumoral , MicroRNAs/genética , MicroRNAs/metabolismo , Proliferação de Células/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Receptores de Netrina/genética , Receptores de Netrina/metabolismo
20.
Cell Biosci ; 13(1): 13, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36670464

RESUMO

BACKGROUND: Atherosclerosis (AS) is the leading underlying cause of the majority of clinical cardiovascular events. Retention of foamy macrophages in plaques is the main factor initiating and promoting the atherosclerotic process. Our previous work showed that ox-LDL induced macrophage retention in plaques and that the guidance receptor Uncoordinated-5 homolog B (Unc5b) was involved in this process. However, little is known about the role of Unc5b in regulating macrophage accumulation within plaques. RESULTS: In the present study, we found that Unc5b controls macrophage migration and thus promotes plaque progression in ApoE-/- mice. The immunofluorescence colocalization assay results first suggested that fucosyltransferase 8 (Fut8) might participate in the exacerbation of atherosclerosis. Animals with Unc5b overexpression showed elevated levels of Fut8 and numbers of macrophages and an increased lesion size and intimal thickness. However, these effects were reversed in ApoE-/- mice with Unc5b knockdown. Furthermore, Raw264.7 macrophages with siRNA-mediated silencing of Unc5b or overexpression of Unc5b were used to confirm the regulatory mechanisms of Unc5b and Fut8 in vitro. In response to ox-LDL exposure, Unc5b and Fut8 were both upregulated, and macrophages showed reduced pseudopod formation and migratory capacities. However, these capacities were restored by blocking Unc5b or Fut8. Furthermore, the IP assay indicated that Fut8 regulated the level of α-1,6 fucosylation of Unc5b, which mainly occurs in the endoplasmic reticulum (ER), and genetic deletion of the main fucosylation sites or Fut8 resulted in hypofucosylation of Unc5b. Moreover, the macrophage migration mediated by Unc5b depended on inactivation of the p-CDC42/p-PAK pathway. Conversely, macrophages with Unc5b overexpression displayed activation of the p-CDC42/p-PAK pathway and decreased migration both in vivo and in vitro. CONCLUSION: These results demonstrated that hypofucosylation of Unc5b regulated by Fut8 is positively associated with the delay of the atherosclerotic process by promoting the migration of foamy macrophages. These findings identify a promising therapeutic target for atherosclerosis.

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