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1.
PLoS One ; 15(7): e0236348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735560

RESUMO

Vocal folds are a viscoelastic multilayered structure responsible for voice production. Vocal fold epithelial damage may weaken the protection of deeper layers of lamina propria and thyroarytenoid muscle and impair voice production. Systemic dehydration can adversely affect vocal function by creating suboptimal biomechanical conditions for vocal fold vibration. However, the molecular pathobiology of systemically dehydrated vocal folds is poorly understood. We used an in vivo rabbit model to investigate the complete gene expression profile of systemically dehydrated vocal folds. The RNA-Seq based transcriptome revealed 203 differentially expressed (DE) vocal fold genes due to systemic dehydration. Interestingly, function enrichment analysis showed downregulation of genes involved in cell adhesion, cell junction, inflammation, and upregulation of genes involved in cell proliferation. RT-qPCR validation was performed for a subset of DE genes and confirmed the downregulation of DSG1, CDH3, NECTIN1, SDC1, S100A9, SPINK5, ECM1, IL1A, and IL36A genes. In addition, the upregulation of the transcription factor NR4A3 gene involved in epithelial cell proliferation was validated. Taken together, these results suggest an alteration of the vocal fold epithelial barrier independent of inflammation, which could indicate a disruption and remodeling of the epithelial barrier integrity. This transcriptome provides a first global picture of the molecular changes in vocal fold tissue in response to systemic dehydration. The alterations observed at the transcriptional level help to understand the pathobiology of dehydration in voice function and highlight the benefits of hydration in voice therapy.


Assuntos
Desidratação/genética , Músculos Laríngeos/metabolismo , Prega Vocal/metabolismo , Distúrbios da Voz/genética , Animais , Fenômenos Biomecânicos , Adesão Celular/genética , Proliferação de Células/genética , Desidratação/metabolismo , Desidratação/patologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica/genética , Humanos , Junções Intercelulares/genética , Músculos Laríngeos/patologia , Membrana Mucosa/metabolismo , Membrana Mucosa/patologia , Coelhos , Prega Vocal/patologia , Distúrbios da Voz/patologia
2.
Proc Natl Acad Sci U S A ; 117(23): 13056-13065, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32439708

RESUMO

Plasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections.


Assuntos
Antígenos de Protozoários/imunologia , Genes de Protozoários , Malária Vivax/imunologia , Plasmodium vivax/imunologia , Baço/metabolismo , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos de Protozoários/genética , Aotidae , Células CHO , Adesão Celular/genética , Adesão Celular/imunologia , Criança , Cricetulus , Modelos Animais de Doenças , Fibroblastos , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Malária Vivax/sangue , Malária Vivax/parasitologia , Família Multigênica , Papua Nova Guiné , Plasmodium vivax/genética , Baço/citologia , Baço/parasitologia , Esplenectomia , Análise Serial de Tecidos
3.
Proc Natl Acad Sci U S A ; 117(16): 9064-9073, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32273388

RESUMO

The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP's role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.


Assuntos
Neoplasias Encefálicas/patologia , Adesões Focais/patologia , Glioblastoma/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neovascularização Patológica/patologia , Citoesqueleto de Actina/metabolismo , Animais , Encéfalo/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Estudos de Coortes , Progressão da Doença , Feminino , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia Intravital , Camundongos , Microscopia Confocal , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neovascularização Patológica/genética , Imagem com Lapso de Tempo , Vinculina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
PLoS One ; 15(4): e0231752, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32330152

RESUMO

Astrocytes (AC) are the most abundant cells in the central nervous system. In the retina, astrocytes play important roles in the development and integrity of the retinal neurovasculature. Astrocytes dysfunction contributes to pathogenesis of a variety of neurovascular diseases including diabetic retinopathy. Recent studies have demonstrated the expression of Cyp1b1 in the neurovascular cells of the central nervous system including AC. We recently showed retinal AC constitutively express Cyp1b1, and global Cyp1b1-deficiency (Cyp1b1-/-) attenuates retinal ischemia-mediated neovascularization in vivo and the pro-angiogenic activity of retinal vascular cells in vitro. We also demonstrated that Cyp1b1 expression is a key regulator of retinal AC function. However, the underlying mechanisms involved need further investigation. Here we determined changes in the transcriptome profiles of Cyp1b1+/+ and Cyp1b1-/- retinal AC by RNA sequencing. We identified 585 differentially expressed genes, whose pathway enrichment analysis revealed the most significant pathways impacted in Cyp1b1-/- AC. These genes included those of axon guidance, extracellular matrix proteins and their receptors, cancer, cell adhesion molecules, TGF-ß signaling, and the focal adhesion modulation. The expression of a selected set of differentially expressed genes was confirmed by RT-qPCR analysis. To our knowledge, this is the first report of RNAseq investigation of the retinal AC transcriptome and the molecular pathways impacted by Cyp1b1 expression. These results demonstrated an important role for Cyp1b1 expression in the regulation of various retinal AC functions, which are important in neurovascular development and integrity.


Assuntos
Astrócitos/fisiologia , Adesão Celular/genética , Citocromo P-450 CYP1B1/metabolismo , Regulação da Expressão Gênica/fisiologia , Retina/fisiologia , Animais , Movimento Celular/genética , Células Cultivadas , Citocromo P-450 CYP1B1/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Cultura Primária de Células , RNA-Seq , Retina/citologia
5.
Proc Natl Acad Sci U S A ; 117(14): 8013-8021, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193335

RESUMO

AMP-activated protein kinase (AMPK) functions as an energy sensor and is pivotal in maintaining cellular metabolic homeostasis. Numerous studies have shown that down-regulation of AMPK kinase activity or protein stability not only lead to abnormality of metabolism but also contribute to tumor development. However, whether transcription regulation of AMPK plays a critical role in cancer metastasis remains unknown. In this study, we demonstrate that AMPKα1 expression is down-regulated in advanced human breast cancer and is associated with poor clinical outcomes. Transcription of AMPKα1 is inhibited on activation of PI3K and HER2 through ΔNp63α. Ablation of AMPKα1 expression or inhibition of AMPK kinase activity leads to disruption of E-cadherin-mediated cell-cell adhesion in vitro and increased tumor metastasis in vivo. Furthermore, restoration of AMPKα1 expression significantly rescues PI3K/HER2-induced disruption of cell-cell adhesion, cell invasion, and cancer metastasis. Together, these results demonstrate that the transcription control is another layer of AMPK regulation and suggest a critical role for AMPK in regulating cell-cell adhesion and cancer metastasis.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Mama/patologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Cromonas/farmacologia , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Lapatinib/farmacologia , Camundongos , Morfolinas/farmacologia , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Prognóstico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Análise Serial de Tecidos , Transcrição Genética/efeitos dos fármacos , Ativação Transcricional , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Nat Immunol ; 21(3): 261-273, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066955

RESUMO

Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Here, we investigate how transcriptional changes in bone marrow (BM) MSCs result in long-lasting effects on HSCs. Single-cell analysis of Cxcl12-abundant reticular (CAR) cells and PDGFRα+Sca1+ (PαS) cells revealed an extensive cellular heterogeneity but uniform expression of the transcription factor gene Ebf1. Conditional deletion of Ebf1 in these MSCs altered their cellular composition, chromatin structure and gene expression profiles, including the reduced expression of adhesion-related genes. Functionally, the stromal-specific Ebf1 inactivation results in impaired adhesion of HSCs, leading to reduced quiescence and diminished myeloid output. Most notably, HSCs residing in the Ebf1-deficient niche underwent changes in their cellular composition and chromatin structure that persist in serial transplantations. Thus, genetic alterations in the BM niche lead to long-term functional changes of HSCs.


Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Transativadores/deficiência , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Autorrenovação Celular/genética , Autorrenovação Celular/fisiologia , Cromatina/genética , Feminino , Hematopoese/genética , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Célula Única , Nicho de Células-Tronco/genética , Nicho de Células-Tronco/fisiologia , Transativadores/genética , Transcriptoma
7.
Mol Carcinog ; 59(4): 339-352, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31989722

RESUMO

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that the alter of the cellular cytoskeleton and intercellular adhesion. CD2-associated protein (CD2AP) is one of the critical proteins regulating cytoskeleton assembly and intercellular adhesion. However, no study has investigated the expression and biological significance of CD2AP in gastric cancer (GC) to date. Therefore, the aim of our study was to explore if the expression of CD2AP is associated with any clinical features of GC and to elucidate the underlying mechanism. Immunohistochemistry of 620 patient tissue samples indicated that the expression of CD2AP is downregulated in DGC. Moreover, a low CD2AP level was indicative of poor patient prognosis. In vitro, forced expression of CD2AP caused a significant decrease in the migration and invasion of GC cells, whereas depletion of CD2AP had the opposite effect. Immunofluorescence analysis indicated that CD2AP promoted cellular adhesion and influenced cell cytoskeleton assembly via interaction with the F-actin capping protein CAPZA1. Overall, the upregulation of CD2AP could attenuate GC metastasis, suggesting CD2AP as a novel biomarker for the prognosis and treatment of patients with GC.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Movimento Celular/genética , Proteínas do Citoesqueleto/genética , Citoesqueleto/metabolismo , Neoplasias Gástricas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Interferência de RNA , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
8.
Sci Adv ; 6(1): eaau5670, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31921998

RESUMO

Directional cell motility relies on the ability of single cells to establish a front-rear polarity and can occur in the absence of external cues. The initiation of migration has often been attributed to the spontaneous polarization of cytoskeleton components, while the spatiotemporal evolution of cell-substrate interaction forces has yet to be resolved. Here, we establish a one-dimensional microfabricated migration assay that mimics the complex in vivo fibrillar environment while being compatible with high-resolution force measurements, quantitative microscopy, and optogenetics. Quantification of morphometric and mechanical parameters of NIH-3T3 fibroblasts and RPE1 epithelial cells reveals a generic stick-slip behavior initiated by contractility-dependent stochastic detachment of adhesive contacts at one side of the cell, which is sufficient to trigger cell motility in 1D in the absence of pre-established polarity. A theoretical model validates the crucial role of adhesion dynamics, proposing that front-rear polarity can emerge independently of a complex self-polarizing system.


Assuntos
Adesão Celular/genética , Movimento Celular/genética , Polaridade Celular/genética , Citoesqueleto/genética , Animais , Comunicação Celular/genética , Simulação por Computador , Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células NIH 3T3
9.
Biomolecules ; 10(2)2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31991559

RESUMO

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase with key roles in the regulation of cell adhesion migration, proliferation and survival. In cancer FAK is a major driver of invasion and metastasis and its upregulation is associated with poor patient prognosis. FAK is autoinhibited in the cytosol, but activated upon localisation into a protein complex, known as focal adhesion complex. This complex forms upon cell adhesion to the extracellular matrix (ECM) at the cytoplasmic side of the plasma membrane at sites of ECM attachment. FAK is anchored to the complex via multiple sites, including direct interactions with specific membrane lipids and connector proteins that attach focal adhesions to the actin cytoskeleton. In migrating cells, the contraction of actomyosin stress fibres attached to the focal adhesion complex apply a force to the complex, which is likely transmitted to the FAK protein, causing stretching of the FAK molecule. In this review we discuss the current knowledge of the FAK structure and how specific structural features are involved in the regulation of FAK signalling. We focus on two major regulatory mechanisms known to contribute to FAK activation, namely interactions with membrane lipids and stretching forces applied to FAK, and discuss how they might induce structural changes that facilitate FAK activation.


Assuntos
Adesão Celular/genética , Quinase 1 de Adesão Focal/genética , Adesões Focais/genética , Mecanotransdução Celular/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Movimento Celular , Quinase 1 de Adesão Focal/ultraestrutura , Adesões Focais/ultraestrutura , Humanos , Membranas/ultraestrutura , Fosforilação , Transdução de Sinais/genética
10.
RNA ; 26(3): 306-323, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900330

RESUMO

MicroRNA (miRNA)-mediated regulation is widespread, relatively mild but functionally important. It remains challenging to unequivocally identify miRNA targeted RNAs at a genomic scale and determine how changes in miRNA levels affect the transcriptome. Here, we captured individual miRNAs and their targeted RNA sites in wild-type, miR-200 family knockout and induced epithelial cells. We detected 1797 miRNAs interacting with 13,830 transcripts at 616,127 sites by sequencing 1,230,019 unique miRNA:RNA chimeras. Although mRNA sites that are bound by miRNAs and contain matches to seed sequences confer the strongest regulation, ∼40%-60% of miRNA bound regions do not contain seed matches. Different miRNAs have different preferences to seed matches and 3' end base-pairing. For individual miRNAs, the effectiveness of mRNA regulation is highly correlated with the number of captured miRNA:mRNA chimeras. Notably, elevated miR-200 expression robustly represses existing targets with little impact on newly recognized targets. Global analysis of directly captured mRNA targets reveals pathways that are involved in cancer and cell adhesion and signaling pathways that are highly regulated by many different miRNAs in epithelial cells. Comparison between experimentally captured and TargetScan predicted targets indicates that our approach is more effective in identifying bona fide targets by reducing false positive and negative predictions. This study reveals the global binding landscape and impact of miRNAs on the mammalian transcriptome.


Assuntos
MicroRNAs/genética , Neoplasias/genética , RNA Mensageiro/genética , Transcriptoma/genética , Animais , Adesão Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Humanos , Mamíferos , Transdução de Sinais/genética
11.
PLoS One ; 15(1): e0227425, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910229

RESUMO

Shigella species cause bacillary dysentery, especially among young individuals. Shigellae target the human colon for invasion; however, the initial adhesion mechanism is poorly understood. The Shigella surface protein IcsA, in addition to its role in actin-based motility, acts as a host cell adhesin through unknown mechanism(s). Here we confirmed the role of IcsA in cell adhesion and defined the region required for IcsA adhesin activity. Purified IcsA passenger domain was able block S. flexneri adherence and was also used as a molecular probe that recognised multiple components from host cells. The region within IcsA's functional passenger domain (aa 138-148) was identified by mutagenesis. Upon the deletion of this region, the purified IcsAΔ138-148 was found to no longer block S. flexneri adherence and had reduced ability to interact with host molecules. Furthermore, S. flexneri expressing IcsAΔ138-148 was found to be significantly defective in both cell adherence and invasion. Taken together, our data identify an adherence region within the IcsA functional domain and provides useful information for designing therapeutics for Shigella infection.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Disenteria Bacilar/genética , Interações entre Hospedeiro e Microrganismos/genética , Shigella flexneri/genética , Fatores de Transcrição/genética , Adesinas Bacterianas/genética , Adesão Celular/genética , Colo/microbiologia , Disenteria Bacilar/microbiologia , Humanos , Mutagênese , Mutação , Shigella flexneri/patogenicidade
12.
J Biomed Sci ; 27(1): 2, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31898491

RESUMO

BACKGROUND: Serglycin (SRGN), previously recognized as an intracellular proteoglycan involved in the storage processes of secretory granules, has recently been shown to be upregulated in several solid tumors. We have previously shown that SRGN in non-small cell lung cancer (NSCLC) promotes malignant phenotypes in a CD44-dependent manner and increased expression of SRGN predicts poor prognosis of primary lung adenocarcinomas. However, the underlying mechanism remains to be defined. METHODS: Overexpression, knockdown and knockout approaches were performed to assess the role of SRGN in cell motility using wound healing and Boyden chamber migration assays. SRGN devoid of glycosaminoglycan (GAG) modification was produced by site-directed mutagenesis or chondroitinase treatment. Liquid chromatography/tandem mass spectrometry was applied for quantitative analysis of the disaccharide compositions and sulfation extent of SRGN GAGs. Western blot and co-immunoprecipitation analyses were performed to determine the expression and interaction of proteins of interest. Actin cytoskeleton organization was monitored by immunofluorescence staining. RESULTS: SRGN expressed by NSCLC cells is readily secreted to the extracellular matrix in a heavily glycosylated form attached with mainly chondroitin sulfate (CS)-GAG chains, and to a lesser extent with heparin sulfate (HS). The CS-GAG moiety serves as the structural motif for SRGN binding to tumor cell surface CD44 and promotes cell migration. SRGN devoid of CS-GAG modification fails to interact with CD44 and has lost the ability to promote cell migration. SRGN/CD44 interaction promotes focal adhesion turnover via Src-mediated paxillin phosphorylation and disassembly of paxillin/FAK adhesion complex, facilitating cell migration. In support, depletion of Src activity or removal of CS-GAGs efficiently blocks SRGN-mediated Src activation and cell migration. SRGN also promotes cell migration via inducing cytoskeleton reorganization mediated through RAC1 and CDC42 activation accompanied with increased lamellipodia and filopodia formation. CONCLUSIONS: Proteoglycan SRGN promotes NSCLC cell migration via the binding of its GAG motif to CD44. SRGN/CD44 interaction induces Rho-family GTPase-mediated cytoskeleton reorganization and facilitates Src-mediated focal adhesion turnover, leading to increased cell migration. These findings suggest that targeting specific glycans in tumor microenvironment that serve as ligands for oncogenic pathways may be a potential strategy for cancer therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Glicosaminoglicanos/genética , Receptores de Hialuronatos/genética , Proteoglicanas/genética , Proteínas de Transporte Vesicular/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Quinase 1 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Glicosaminoglicanos/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Proteoglicanas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rho de Ligação ao GTP/genética , Quinases da Família src/genética
13.
Chem Biol Interact ; 315: 108900, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31738905

RESUMO

Synthetic amorphous silica nanoparticles (SAS) are used widely in industrial applications. These nanoparticles are not classified for their carcinogenicity in humans. However, some data still demonstrate a potential carcinogenic risk of these compounds in humans. The Bhas 42 cell line was developed to screen chemicals, as tumor-initiators or -promoters according to their ability to trigger cell-to-cell transformation, in a cell transformation assay. In the present study, we performed unsupervised transcriptomic analysis after exposure of Bhas 42 cells to NM-203 SAS as well as to positive (Min-U-Sil 5® crystalline silica microparticles, and 12-O-tetradecanoylphorbol-13-acetate) and negative (diatomaceous earth) control compounds. We identified a common gene signature for 21 genes involved in the early stage of the SAS- Min-U-Sil 5®- or TPA-induced cell transformation. These genes were related to cell proliferation (over expression) and cell adhesion (under expression). Among them, 12 were selected on the basis of their potential impact on cell transformation. RT-qPCR and western blotting were used to confirm the transcriptomic data. Moreover, similar gene alterations were found when Bhas 42 cells were treated with two other transforming SAS. In conclusion, the results obtained in the current study highlight a 12-gene signature that could be considered as a potential early "bio-marker" of cell transformation induced by SAS and perhaps other chemicals.


Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Nanopartículas/administração & dosagem , Dióxido de Silício/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Biomarcadores Tumorais/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Camundongos , Transcriptoma/genética
14.
Microbiol Res ; 231: 126351, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31707298

RESUMO

The ability of yeast to adhere to biotic and abiotic surfaces represents an essential trait during the early stages of infection. Agglutinin-like sequence (Als) cell-wall proteins play a key role in adhesion of Candida species. Candida parapsilosis genome encompasses 5 ALS members, of which only the role of CPAR2_404800 has been elucidated. The present project was aimed at investigating the contribution of C. parapsilosis Als proteins by generating edited strains lacking functional Als proteins. CPAR2_404770 and CPAR2_404780, further indicated as CpALS4770 and CpALS4780, were selected for the generation of single and double edited strains using an episomal CRISPR/Cas9 technology. Phenotypic characterization of mutant strains revealed that editing of both genes had no impact on the in vitro growth of C. parapsilosis or on morphogenesis. Notably, CpALS4770-edited strain showed a reduction of biofilm formation and adhesive properties to human buccal cells (HBECs). Conversely, single CpALS4780-edited strain did not show any difference compared to the wild-type strain in all the assays performed, while the double CpALS4770-CpALS4780 mutant revealed an increased ability to produce biofilm, a hyper-adhesive phenotype to HBECs, and a marked tendency to form cellular aggregates. Murine vaginal infection experiments indicated a significant reduction in CFUs recovered from BALC/c mice infected with single and double edited strains, compared to those infected with the wild-type strain. These finding clearly indicate that CpAls4770 plays a role in adhesion to biotic and abiotic surfaces, while both CpALS4770 and CpALS4780 genes are required for C. parapsilosis ability to colonize and persist in the vaginal mucosa.


Assuntos
Candida parapsilosis , Adesão Celular/genética , Virulência/genética , Animais , Biofilmes/crescimento & desenvolvimento , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Candida parapsilosis/genética , Candida parapsilosis/patogenicidade , Candidíase , Técnicas de Cultura de Células , Feminino , Proteínas Fúngicas/genética , Inativação Gênica , Genes Fúngicos , Humanos , Camundongos , Membrana Mucosa/microbiologia
15.
Int J Cancer ; 146(6): 1553-1567, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31503345

RESUMO

Detachment of cancer cells from the primary tumor and formation of spheroids in ascites is required for implantation metastasis in epithelial ovarian cancer (EOC), but the underlying mechanism of this process has not been thoroughly elucidated. To mimic this process, ovarian cancer cells were grown in 3D and 2D culture. Hey and OVCA433 spheroids exhibited decreased cell proliferation and enhanced adhesion and invasion. SMYD3 expression was elevated in ovarian carcinoma spheroids in association with increased H3K4 methylation. Depletion of SMYD3 by transient siRNA, stable shRNA knockdown and the SMYD3 inhibitor BCI-121 all decreased spheroid invasion and adhesion. Gene expression arrays revealed downregulation of integrin family members. Inhibition assays confirmed that invasion and adhesion of spheroids are mediated by ITGB6 and ITGAM. SMYD3-deficient cells regained the ability to invade and adhere after forced overexpression of SMYD3, ITGB6 and ITGAM. However, this biological ability was not restored by forced overexpression of SMYD3 in ITGB6- and/or ITGAM-deficient cancer cells. SMYD3 and H3K4me3 binding at the ITGB6 and ITGAM promoters was increased in spheroids compared to that in monolayer cells, and the binding was decreased when SMYD3 expression was inhibited, consistent with the expression changes in integrins. SMYD3 expression and integrin-mediated adhesion were also activated in an intraperitoneal xenograft model and in EOC patient spheroids. In vivo, SMYD3 knockdown inhibited tumor metastasis and reduced ascites volume in both the intraperitoneal xenograft model and a PDX model. Overall, our results suggest that the SMYD3-H3K4me3-integrin pathway plays a crucial role in ovarian cancer metastasis to the peritoneal surface.


Assuntos
Ascite/patologia , Carcinoma Epitelial do Ovário/secundário , Histona-Lisina N-Metiltransferase/metabolismo , Integrinas/genética , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/secundário , Adulto , Idoso , Ascite/etiologia , Carcinoma Epitelial do Ovário/genética , Adesão Celular/genética , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Metilação de DNA , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Peritoneais/genética , Regiões Promotoras Genéticas/genética , Esferoides Celulares , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Biochim Biophys Acta Gen Subj ; 1864(1): 129450, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676296

RESUMO

BACKGROUND: Leucine rich Aspartate motifs (LD motifs) are molecular recognition motifs on Paxillin that recognize LD-motif binding domains (LDBD) of a number of focal adhesion proteins in order to carry out downstream signaling and actin cytoskeleton remodeling. In this study, we identified structural features within LDBDs that influence their binding affinity with Paxillin LD motifs. METHODS: Various point mutants of focal adhesion targeting (FAT) domain of Focal Adhesion Kinase (FAK) were created by moving a key Lysine residue two and three helical turns in order to match the unique conformations as observed in LDBDs of two other focal adhesion proteins, Vinculin and CCM3. RESULTS: This led to identify a mutant of FAT domain of FAK, named as FAT(NV) (Asn992 of FAT domain was replaced by Val), with remarkable high affinity for LD1 (Kd = 1.5 µM vs no-binding with wild type) and LD2 peptides (Kd = 7.2 µM vs 63 µM with wild type). Consistently, the focal adhesions of MCF7 cells expressing FAK(NV) were highly stable (turnover rate = 1.25 × 10-5 µm2/s) as compared to wild type FAK transfected cells (turnover rate = 1.5 × 10-3 µm2/s). CONCLUSIONS: We observed that the relative disposition of key LD binding amino-acids at LDBD surface, hydrophobic burial of long Leucine side chains of LD-motifs and complementarity of charged surfaces are the key factors determining the binding affinities of LD motifs with LDBDs. GENERAL SIGNIFICANCE: Our study will help in protein engineering of FAT domain of FAK by modulating FAK-LD motif interactions which have implications in cellular focal adhesions and cell migration.


Assuntos
Adesão Celular/genética , Quinase 1 de Adesão Focal/genética , Adesões Focais/genética , Conformação Proteica , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Ácido Aspártico/genética , Sítios de Ligação/genética , Movimento Celular/genética , Quinase 1 de Adesão Focal/química , Adesões Focais/química , Regulação da Expressão Gênica/genética , Humanos , Lisina/química , Lisina/genética , Células MCF-7 , Proteínas de Membrana/química , Proteínas de Membrana/genética , Paxilina/química , Paxilina/genética , Ligação Proteica/genética , Engenharia de Proteínas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Vinculina/química , Vinculina/genética
17.
Biochim Biophys Acta Gen Subj ; 1864(1): 129449, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678146

RESUMO

BACKGROUND: Galectins are multifunctional effectors, which all share absence of a signal sequence. It is not clear why galectins belong to the small set of proteins, which avoid the classical export route. METHODS: Products of recombinant galectin expression in P. pastoris were analyzed by haemagglutination, gel filtration and electrophoresis and lectin blotting as well as mass spectrometry on the level of tryptic peptides and purified glycopeptides(s). Density gradient centrifugation and confocal laser scanning microscopy facilitated localization in transfected human and rat cells, proliferation assays determined activity as growth mediator. RESULTS: Directing galectin-1 to the classical secretory pathway in yeast produces N-glycosylated protein that is active. It cofractionates and -localizes with calnexin in human cells, only Gal-4 is secreted. Presence of N-glycan(s) reduces affinity of cell binding and growth regulation by Gal-1. CONCLUSIONS: Folding and activity of a galectin are maintained in signal-peptide-directed routing, N-glycosylation occurs. This pathway would deplete cytoplasm and nucleus of galectin, presence of N-glycans appears to interfere with lattice formation. GENERAL SIGNIFICANCE: Availability of glycosylated galectins facilitates functional assays to contribute to explain why galectins invariably avoid classical routing for export.


Assuntos
Adesão Celular/genética , Galectina 1/genética , Galectina 4/genética , Sinais Direcionadores de Proteínas/genética , Animais , Transporte Biológico , Calnexina/genética , Linhagem Celular , Galectina 1/química , Galectina 4/química , Glicosilação , Humanos , Polissacarídeos/química , Polissacarídeos/genética , Dobramento de Proteína , Ratos , Transdução de Sinais/genética
18.
J Cell Physiol ; 235(1): 454-464, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31264215

RESUMO

Lung adenocarcinomas injured greatly on the people worldwide. Although clinic experiments and gene profiling analyses had been well performed, to our knowledge, systemic coexpression analysis of human genes for this cancer is still limited to date. Here, using the published data GSE75037, we built the coexpression modules of genes by Weighted Gene Co-Expression Network Analysis (WGCNA), and investigated function and protein-protein interaction network of coexpression genes by Database for Annotation, visualization, and Integrated Discovery (DAVID) and String database, respectively. First, 11 coexpression modules were conducted for 5,000 genes in the 83 samples recently. Number of genes for each module ranged from 90 to 1,260, with the mean of 454. Second, interaction relationships of hub-genes between pairwise modules showed great differences, suggesting relatively high scale independence of the modules. Third, functional enrichment of the coexpression modules showed great differences. We found that genes in modules 8 significantly enriched in the biological process and/or pathways of cell adhesion, extracellular matrix (ECM)-receptor interaction, focal adhesion, and PI3K-Akt signaling pathway, and so forth. It was inferred as the key module underlying lung adenocarcinomas. Furthermore, PPI analysis revealed that the genes COL1A1, COL1A2, COL3A1, CTGF, and BGN owned the largest number of adjacency genes, unveiling that they may functioned importantly during the occurrence of lung adenocarcinomas. To summary, genes involved in cell adhesion, ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathway play crucial roles in human lung adenocarcinomas.


Assuntos
Adenocarcinoma de Pulmão/genética , Adesão Celular/genética , Matriz Extracelular/metabolismo , Adesões Focais/genética , Neoplasias Pulmonares/genética , Adenocarcinoma de Pulmão/patologia , Biglicano/genética , Colágeno Tipo I/genética , Colágeno Tipo III/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes , Humanos , Neoplasias Pulmonares/patologia , Fosfatidilinositol 3-Quinases/genética , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Transcriptoma/genética
19.
Med Sci Monit ; 25: 9227-9236, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31796725

RESUMO

BACKGROUND The purpose of this study was to investigate whether Orai1 plays a role in the metastasis of osteosarcoma. MATERIAL AND METHODS The expression of Orai1 was silenced by small interfering RNAs against Orai1 (Orai1 siRNA) in osteosarcoma MG-63 cells. Various experiments were carried out to detect the changes in migration, invasion, and adhesion ability of these osteosarcoma cells. Furthermore, the activity of Rac1, Wave2, and Ras was detected using Western blot analysis. Moreover, the Rac1 and Ras inhibitors were used to confirm whether the Ras-Rac1-WAVE2 signaling pathway was involved in osteosarcoma metastasis promoted by Orai1. RESULTS We found that the migration, invasion, and adhesion ability of MG-63 cells were significantly reduced after silencing Orai1 expression (p<0.05). Moreover, the activity of the Rac1-WAVE2 signaling pathway was significantly inhibited after silencing of Orai1 expression (p<0.05). After the Rac1 inhibitor was added, Orai1 siRNA could not further inhibit migration, invasion, and adhesion of the osteosarcoma cells. Further experiments showed that Ras activity was significantly inhibited after silencing Orai1 expression (p<0.05). Moreover, Orai1 siRNA did not further inhibit the activity of the Rac1-WAVE2 signaling pathway nor did it further inhibit the migration, invasion, and adhesion ability of osteosarcoma cells following the addition of Ras inhibitors. CONCLUSIONS Orai1 activates the Ras-Rac1-WAVE2 signaling pathway to promote metastasis of osteosarcoma. Abnormal expression or function of Orai1 may be an important cause of osteosarcoma metastasis.


Assuntos
Neoplasias Ósseas/metabolismo , Proteína ORAI1/metabolismo , Osteossarcoma/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Proteína ORAI1/genética , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/patologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas ras/genética
20.
Acta Biochim Pol ; 66(4): 499-507, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31883363

RESUMO

The MSCs are immature cells that can be found in numerous different tissue types. In recent years, they have gained considerable attention, particularly with regard to their regenerative properties. Due to their paracrine activity, ability to migrate, adhesion and homing, MSCs currently appear to be the most relevant for therapeutic use. Numerous bioactive molecules secreted by MSCs exert paracrine effects and modulate many physiological processes, such as angiogenesis, immunomodulation and neuroprotection. Cell-cell communication may be also mediated by extracellular vesicles released from the cells. Due to these properties, MSCs have been widely studied for evaluation of their therapeutic benefits expected in the clinical applications. For effective tissue regeneration, transplanted MSCs have to exit the circulation and locate at the site of damage, which is possible because of their ability to migrate, adhere and engraft at the target site. Accumulating evidence suggests that MSCs recruitment from remote sites is similar to leukocytes' migration. All of these biological features make MSCs highly investigated stem cells and the most commonly used cells in regenerative medicine. Since environmental factors affect the MSCs behavior, we discuss importance of oxygen concentration as a one of the key factors affecting MSCs properties.


Assuntos
Adesão Celular/genética , Transplante de Células-Tronco Mesenquimais , Neovascularização Patológica/terapia , Medicina Regenerativa , Comunicação Celular/genética , Movimento Celular/genética , Humanos , Leucócitos/citologia , Células-Tronco Mesenquimais/citologia
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