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1.
Methods Mol Biol ; 2217: 71-81, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33215378

RESUMO

The in situ proximity ligation assay (PLA) is capable of detecting single protein events such as protein protein-interactions and posttranslational modifications (e.g., protein phosphorylation) in tissue and cell samples prepared for analysis by immunofluorescent or immunohistochemical microscopy. The targets are detected using two primary antibodies which must be from different host species. A pair of secondary antibodies (PLA probes) conjugated to complementary oligonucleotides is applied to the sample, and a signal is generated only when the two PLA probes are in close proximity by their binding to the two primary antibodies that have bound to their targets in close proximity. The signal from each pair of PLA probes is visualized as an individual fluorescent spot. These PLA signals can be quantified (counted) using image analysis software (ImageJ), and also assigned to a specific subcellular location based on microscopy image overlays. In principle, in situ PLA offers a relatively simple and sensitive technique to analyze interactions among any proteins for which suitable antibodies are available. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes consisting of more than 150 proteins, also known as the integrin adhesome, which link the extracellular matrix (ECM) to the actin cytoskeleton and regulate the functioning of mechanosignaling pathways. The in situ PLA approach is well suited for examining the spatiotemporal aspects of protein posttranslational modifications and protein interactions occurring in dynamic multiprotein complexes such as integrin mediated focal adhesions.


Assuntos
Adesões Focais/metabolismo , Imuno-Histoquímica/métodos , Cadeias alfa de Integrinas/metabolismo , Integrina beta1/metabolismo , Complexos Multiproteicos/metabolismo , Oligonucleotídeos/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Anticorpos/química , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Adesões Focais/ultraestrutura , Mucosa Gástrica/metabolismo , Mucosa Gástrica/ultraestrutura , Humanos , Processamento de Imagem Assistida por Computador , Cadeias alfa de Integrinas/química , Integrina beta1/química , Microscopia de Fluorescência , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Complexos Multiproteicos/química , Músculo Liso/metabolismo , Músculo Liso/ultraestrutura , Oligonucleotídeos/síntese química , Ligação Proteica
2.
Am J Physiol Heart Circ Physiol ; 320(2): H734-H739, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33337960

RESUMO

The integrin family, an indispensable part of cell-cell and cell-matrix interactions, consists of a group of heterodimeric adhesion receptors formed by α- and ß-integrin subunits. Their wide expression and unique bidirectional signaling pathways allow them to play roles in a variety of biological activities including blood clot formation, cell attachment, and migration. Evidence suggests that integrins are essential regulators of the initiation of acute inflammation, especially two key aspects of this process i.e., vascular permeability and leukocyte recruitment. This mini-review discusses the importance of integrins at the onset of the acute inflammatory response and outlines research advances regarding the function of integrins and their modulators at different stages of this process. Insights into the fine-tuning of integrin signaling during acute inflammation may inspire the design of new drugs for inflammatory diseases.


Assuntos
Antígenos CD18/metabolismo , Permeabilidade Capilar , Quimiotaxia de Leucócito , Endotélio Vascular/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Integrina beta1/metabolismo , Leucócitos/metabolismo , Animais , Adesão Celular , Comunicação Celular , Endotélio Vascular/imunologia , Endotélio Vascular/fisiopatologia , Humanos , Inflamação/imunologia , Inflamação/fisiopatologia , Migração e Rolagem de Leucócitos , Leucócitos/imunologia , Transdução de Sinais , Migração Transendotelial e Transepitelial
3.
Fa Yi Xue Za Zhi ; 36(4): 502-506, 2020 Aug.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-33047534

RESUMO

Abstract: Objective To study the characteristics of positive expression of integrin ß1 in the rat brain tissue of two kinds of traumatic brain injury models and to explore the feasibility of inferring the mode of traumatic brain injury using the positive expression of integrin ß1. Methods The occipital region of rats was hit by hydraulic impact method and pendulum striking method to produce two closed brain injury models of linear and rotation acceleration respectively, then 120 SD rats were randomly divided into linear acceleration injury group, rotation acceleration injury group, sham operation group and normal control group. Immunohistochemistry staining and Western blotting method were used to detect the positive expression of integrin ß1 in different parts of the brain tissue at 30 min, 3 h, 6 h, 12 h, 3 d and 7 d after rat injury. The data was processed statistically by SPSS 18.0 software. Results The positive expression of integrin ß1 was detected 30 min after brain injury and reached the peak 6 h after brain injury. With the extension of injury time, the expression tended to enhance. At the same time points after injury, the differences in the positive expression of integrin ß1 between the linear acceleration injury group and the rotation acceleration injury group in the occipital strike point and thalamus had no statistical significance ( P>0.05), but the differences in the expression of integrin ß1 in the frontal lobe and brain stem had statistical significance (P<0.05). Conclusion The characteristics of positive expression of integrin ß1 in brain tissue can be used to infer the strike point and the manner of injury and has application value for the reconstruction of craniocerebral injury process.


Assuntos
Lesões Encefálicas Traumáticas , Lesões Encefálicas , Animais , Encéfalo/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Ratos , Ratos Sprague-Dawley
4.
Am J Physiol Renal Physiol ; 319(4): F603-F611, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32830538

RESUMO

The acyl-CoA synthetase medium-chain family member 2 (Acsm2) gene was first identified and cloned by our group as a kidney-specific "KS" gene. However, its expression pattern and function remain to be clarified. In the present study, we found that the Acsm2 gene was expressed specifically and at a high level in normal adult kidneys. Expression of Acsm2 in kidneys followed a maturational pattern: it was low in newborn mice and increased with kidney development and maturation. In situ hybridization and immunohistochemistry revealed that Acsm2 was expressed specifically in proximal tubular cells of adult kidneys. Data from the Encyclopedia of DNA Elements database revealed that the Acsm2 gene locus in the mouse has specific histone modifications related to the active transcription of the gene exclusively in kidney cells. Following acute kidney injury, partial unilateral ureteral obstruction, and chronic kidney diseases, expression of Acsm2 in the proximal tubules was significantly decreased. In human samples, the expression pattern of ACSM2A, a homolog of mouse Acsm2, was similar to that in mice, and its expression decreased with several types of renal injuries. These results indicate that the expression of Acsm2 parallels the structural and functional maturation of proximal tubular cells. Downregulation of its expression in several models of kidney disease suggests that Acms2 may serve as a novel marker of proximal tubular injury and/or dysfunction.


Assuntos
Coenzima A Ligases/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais Proximais/metabolismo , Lesão Renal Aguda/enzimologia , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Coenzima A Ligases/genética , Modelos Animais de Doenças , Células Epiteliais/patologia , Fibrose , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Insuficiência Renal Crônica/enzimologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Renina/genética , Renina/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia
5.
Medicine (Baltimore) ; 99(27): e20944, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32629699

RESUMO

BACKGROUND: Colorectal cancer is the second commonly seen cancer around the world and accounts for 13% of all human cancers. Among them, 25% of all case were diagnosed with metastasis and 50% occurs metastasis during the development of disease. Cetuximab is a chimeric monoclonal antibody against epidermal growth factor receptor, and is used for treatment of metastatic colorectal cancer alone or combined with chemotherapy or radiation therapy. Integrin-beta 1 (ITGB1), which is also known as CD29, and plays an important role in development of malignant cancers. However, the effect of ITGB1 in promoting the anti-tumor effect of cetuximab is not fully understand. METHODS: The model of ITGB1 inhibition and overexpression was firstly constructed in LS174T cells, and the viability of cells in each group was detected using CCK-8 assay. The expression of key factors in tumor formation process at transcription level was detected using real-time quantitative polymerase chain reaction method. The expression of key proteins in metastasis process, cell apoptosis and activation of Ras/Raf/MEK signaling pathway was detected using western blotting analysis. And the concentration of key factors of in tumor formation process in cultured medium of LS174T cells were detected using enzyme-linked immunosorbent assay method. RESULTS: We found that cetuximab could inhibit the proliferation of LS174T cells, and inhibition of ITGB1 enhanced this effect while overexpression of ITGB1 reduced this effect. We further found that cetuximab could inhibit the expression and secretion of extracellular matrix degradation related molecules in cultured medium and transcription level. Besides, we also found that the expression of key factors in angiogenesis and extracellular matrix degradation related proteins were also reduced after cetuximab treatment. These effects might be mediated by Ras/Raf/MAPK signaling pathway and enhanced after inhibition of ITGB1 expression. CONCLUSION: Inhibition of ITGB1 might be a new therapeutic method in colorectal cancer.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Cetuximab/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Integrina beta1/efeitos dos fármacos , Antineoplásicos Imunológicos/uso terapêutico , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Cetuximab/uso terapêutico , Neoplasias Colorretais/patologia , Humanos , Integrina beta1/metabolismo
6.
PLoS One ; 15(6): e0225173, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32603328

RESUMO

Vascular hyperplasia after vascular trauma is one of the difficult problems in clinical treatment. Nowadays, there is no effective treatment for vascular hyperplasia. Previous studies have shown that integrinß1 andß3 activity play an important role in vascular hyperplasia. Kindlin-2 has been shown to modulate integrinß1 andß3 activity in cancer. Therefore, in this study, we hope to explore the relationship between Kindlin-2 and vascular hyperplasia. We overexpressed or knocked down Kindlin-2 by adenovirus. The results showed that Kindlin-2 overexpression could regulate integrinß1 andß3 activity through FAK-PIK3 signaling pathways ex vivo and in vivo, thereby affecting the proliferation and migration of VSMC, and then it causes the consequences of vascular hyperplasia. Therefore, Our results show that Kindlin-2 may be a potential target for the treatment of vascular hyperplasia.


Assuntos
Movimento Celular , Quinase 1 de Adesão Focal/metabolismo , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Proteínas de Membrana/metabolismo , Músculo Liso Vascular/citologia , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Animais , Proliferação de Células , Técnicas de Silenciamento de Genes , Hiperplasia/metabolismo , Hiperplasia/patologia , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Ratos
7.
Nat Commun ; 11(1): 3017, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541798

RESUMO

Breast cancer brain metastases (BCBM) have a 5-20 year latency and account for 30% of mortality; however, mechanisms governing adaptation to the brain microenvironment remain poorly defined. We combine time-course RNA-sequencing of BCBM development with a Drosophila melanogaster genetic screen, and identify Rab11b as a functional mediator of metastatic adaptation. Proteomic analysis reveals that Rab11b controls the cell surface proteome, recycling proteins required for successful interaction with the microenvironment, including integrin ß1. Rab11b-mediated control of integrin ß1 surface expression allows efficient engagement with the brain ECM, activating mechanotransduction signaling to promote survival. Lipophilic statins prevent membrane association and activity of Rab11b, and we provide proof-of principle that these drugs prevent breast cancer adaptation to the brain microenvironment. Our results identify Rab11b-mediated recycling of integrin ß1 as regulating BCBM, and suggest that the recycleome, recycling-based control of the cell surface proteome, is a previously unknown driver of metastatic adaptation and outgrowth.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/patologia , Integrina beta1/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/fisiopatologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Humanos , Integrina beta1/genética , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Transporte Proteico , Transdução de Sinais , Microambiente Tumoral , Proteínas rab de Ligação ao GTP/genética
8.
Am J Obstet Gynecol ; 223(5): 733.e1-733.e14, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32417359

RESUMO

BACKGROUND: Uterine leiomyomas, the most common tumors of the female reproductive system, are characterized by excessive deposition of disordered stiff extracellular matrix and fundamental alteration in the mechanical signaling pathways. Specifically, these alterations affect the normal dynamic state of responsiveness to mechanical cues in the extracellular environment. These mechanical cues are converted through integrins, cell membrane receptors, to biochemical signals including cytoskeletal signaling pathways to maintain mechanical homeostasis. Leiomyoma cells overexpress ß1 integrin and other downstream mechanical signaling proteins. We previously reported that simvastatin, an antihyperlipidemic drug, has antileiomyoma effects through cellular, animal model, and epidemiologic studies. OBJECTIVE: This study aimed to examine the hypothesis that simvastatin might influence altered mechanotransduction in leiomyoma cells. STUDY DESIGN: This is a laboratory-based experimental study. Primary leiomyoma cells were isolated from 5 patients who underwent hysterectomy at the Department of Gynecology and Obstetrics of the Johns Hopkins University Hospital. Primary and immortalized human uterine leiomyoma cells were treated with simvastatin at increasing concentrations (0.001, 0.01, 0.1, and 1 µM, or control) for 48 hours. Protein and mRNA levels of ß1 integrin and extracellular matrix components involved in mechanical signaling were quantified by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. In addition, we examined the effect of simvastatin on the activity of Ras homolog family member A using pull-down assay and gel contraction. RESULTS: We found that simvastatin significantly reduced the protein expression of ß1 integrin by 44% and type I collagen by 60% compared with untreated leiomyoma cells. Simvastatin-treated cells reduced phosphorylation of focal adhesion kinase down to 26%-60% of control, whereas it increased total focal adhesion kinase protein expression. Using a Ras homolog family member A pull-down activation assay, we observed reduced levels of active Ras homolog family member A in simvastatin-treated cells by 45%-85% compared with control. Consistent with impaired Ras homolog family member A activation, simvastatin treatment reduced tumor gel contraction where gel area was 122%-153% larger than control. Furthermore, simvastatin treatment led to reduced levels of mechanical signaling proteins involved in ß1 integrin downstream signaling, such as A-kinase anchor protein 13, Rho-associated protein kinase 1, myosin light-chain kinase, and cyclin D1. CONCLUSION: The results of this study suggest a possible therapeutic role of simvastatin in restoring the altered state of mechanotransduction signaling in leiomyoma. Collectively, these findings are aligned with previous epidemiologic studies and other reports and support the need for clinical trials.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Leiomioma/genética , Mecanotransdução Celular/efeitos dos fármacos , Sinvastatina/farmacologia , Neoplasias Uterinas/genética , Proteínas de Ancoragem à Quinase A/efeitos dos fármacos , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Ciclina D1/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina beta1/efeitos dos fármacos , Integrina beta1/genética , Integrina beta1/metabolismo , Leiomioma/metabolismo , Mecanotransdução Celular/genética , Antígenos de Histocompatibilidade Menor/efeitos dos fármacos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Quinase de Cadeia Leve de Miosina/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Cultura Primária de Células , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Neoplasias Uterinas/metabolismo , Quinases Associadas a rho/efeitos dos fármacos , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
9.
Hum Cell ; 33(3): 663-675, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32409959

RESUMO

This study aims to investigate how Maspin affects the EMT and angiogenesis of gastric cancer (GC) cells via ITGB1/FAK pathway. Immunohistochemistry was used to evaluate the expressions of Maspin, ITGB1, FAK, E-cadherin, Vimentin, D2-40, and CD34 in GC and adjacent normal tissues from 160 patients. Then, the human GC cells with different degree of differentiation were transfected with Maspin CRISPR activation plasmid, ITGB1 siRNA and/or Maspin siRNA, followed by the following experiments, including qRT-PCR, western blotting, tube formation assay, Transwell assay and wound healing. GC tumor tissues manifested decreased Maspin with the activated ITGB1/FAK pathway. In tumor tissues, Maspin was negatively correlated with the expressions of ITGB1 and FAK, as well as Lauren's classification, differentiation degree, and TNM stage. Besides, Maspin was negatively related with lymphatic vessel density (LVD) and microvessel density (MVD), Vimentin and VEGF, but was positive correlated with E-cadherin. Maspin expression decreased, but ITGB1 and p-FAK expressions increased gradually in MKN-28 (well differentiated), SGC-7901 (moderate differentiated), and MKN-45 (poorly differentiated). Maspin CRISPR and ITGB1 siRNA increased E-cadherin with the decreased Vimentin, VEGF and bFGF, and the reductions of tube length. In comparison with the ITGB1 siRNA group, cells in the Maspin siRNA + ITGB1 siRNA group presented the more evident EMT and angiogenesis. Furthermore, ITGB1 siRNA reduced the malignancies of GC cells, which could be restored by Maspin siRNA. Maspin was downregulated in GC tissues, which could inhibit the EMT and angiogenesis by blocking the ITGB1/FAK pathway, thereby decreasing cell invasion and migration of GC.


Assuntos
Movimento Celular/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Integrina beta1/metabolismo , Neovascularização Patológica/prevenção & controle , Serpinas/farmacologia , Serpinas/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Humanos , Terapia de Alvo Molecular , Invasividade Neoplásica/genética , Serpinas/uso terapêutico
10.
Am J Physiol Renal Physiol ; 318(5): F1306-F1312, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32308017

RESUMO

Defects in the function of primary cilia are commonly associated with the development of renal cysts. On the other hand, the intact cilium appears to contribute a cystogenic signal whose effectors remain unclear. As integrin-ß1 is required for the cystogenesis caused by the deletion of the polycystin 1 gene, we asked whether it would be similarly important in the cystogenetic process caused by other ciliary defects. We addressed this question by investigating the effect of integrin-ß1 deletion in a ciliopathy genetic model in which the Ift88 gene, a component of complex B of intraflagellar transport that is required for the proper assembly of cilia, is specifically ablated in principal cells of the collecting ducts. We showed that the renal cystogenesis caused by loss of Ift88 is prevented when integrin-ß1 is simultaneously depleted. In parallel, pathogenetic manifestations of the disease, such as increased inflammatory infiltrate and fibrosis, were also significantly reduced. Overall, our data indicate that integrin-ß1 is also required for the renal cystogenesis caused by ciliary defects and point to integrin-ß1-controlled pathways as common drivers of the disease and as possible targets to interfere with the cystogenesis caused by ciliary defects.


Assuntos
Cílios/metabolismo , Integrina beta1/metabolismo , Doenças Renais Císticas/metabolismo , Rim/metabolismo , Animais , Aquaporina 2/genética , Aquaporina 2/metabolismo , Cílios/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Fibrose , Mediadores da Inflamação/metabolismo , Integrina beta1/genética , Rim/patologia , Rim/fisiopatologia , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Doenças Renais Císticas/prevenção & controle , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Knockout , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(18): 9896-9905, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32321834

RESUMO

The extracellular matrix (ECM) initiates mechanical cues that activate intracellular signaling through matrix-cell interactions. In blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from the ECM and their interaction with the mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood. Here, we identified the matricellular protein thrombospondin-1 (Thbs1) as an extracellular mediator of matrix mechanotransduction that acts via integrin αvß1 to establish focal adhesions and promotes nuclear shuttling of Yes-associated protein (YAP) in response to high strain of cyclic stretch. Thbs1-mediated YAP activation depends on the small GTPase Rap2 and Hippo pathway and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin ß1/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload and inhibition of neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall. We thus propose a mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Integrina beta1/genética , Trombospondina 1/genética , Fatores de Transcrição/genética , Remodelação Vascular/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Aorta/crescimento & desenvolvimento , Aorta/metabolismo , Artérias Carótidas/crescimento & desenvolvimento , Artérias Carótidas/metabolismo , Microambiente Celular/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Adesões Focais/genética , Humanos , Integrina beta1/metabolismo , Mecanotransdução Celular , Camundongos , Neointima/genética , Neointima/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Trombospondina 1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas rap de Ligação ao GTP/genética
12.
Arterioscler Thromb Vasc Biol ; 40(6): 1464-1478, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32268789

RESUMO

OBJECTIVE: Despite the current antiatherosclerotic and antithrombotic therapies, the incidence of advanced atherosclerosis-associated clinical events remains high. Whether long noncoding RNAs (lncRNAs) affect the progression of atherosclerosis and whether they are potential targets for the treatment of advanced atherosclerosis are poorly understood. Approach and Results: The progression of atherosclerotic lesions was accompanied by dynamic alterations in lncRNA expression, as revealed by RNA sequencing and quantitative polymerase chain reaction. Among the dynamically changing lncRNAs, we identified a novel lncRNA, lncRNA Associated with the Progression and Intervention of Atherosclerosis (RAPIA), that was highly expressed in advanced atherosclerotic lesions and in macrophages. Inhibition of RAPIA in vivo not only repressed the progression of atherosclerosis but also exerted atheroprotective effects similar to those of atorvastatin on advanced atherosclerotic plaques that had already formed. In vitro assays demonstrated that RAPIA promoted proliferation and reduced apoptosis of macrophages. A molecular sponge interaction between RAPIA and microRNA-183-5p was demonstrated by dual-luciferase reporter and RNA immunoprecipitation assays. Rescue assays indicated that RAPIA functioned at least in part by targeting the microRNA-183-5p/ITGB1 (integrin ß1) pathway in macrophages. In addition, the transcription factor FoxO1 (forkhead box O1) could bind to the RAPIA promoter region and facilitate the expression of RAPIA. CONCLUSIONS: The progression of atherosclerotic lesions was accompanied by dynamic changes in the expression of lncRNAs. Inhibition of the pivotal lncRNA RAPIA may be a novel preventive and therapeutic strategy for advanced atherosclerosis, especially in patients resistant or intolerant to statins.


Assuntos
Aterosclerose/terapia , Expressão Gênica , Macrófagos/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , Animais , Apoptose/efeitos dos fármacos , Aterosclerose/genética , Aterosclerose/prevenção & controle , Atorvastatina/farmacologia , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Proteína Forkhead Box O1/metabolismo , Humanos , Integrina beta1/metabolismo , Macrófagos/química , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , MicroRNAs/metabolismo , MicroRNAs/farmacologia , Regiões Promotoras Genéticas/fisiologia , Células RAW 264.7 , RNA Longo não Codificante/fisiologia
13.
Arch Med Res ; 51(2): 115-123, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32111497

RESUMO

BACKGROUND AND AIMS: Long noncoding RNAs have been proved to play a key role in the development and progression of various tumors, including osteosarcoma (OS). However, the role and molecular mechanism of lncRNA in osteosarcoma metastasis remains unknown. Our purpose is to explore the clinical significance and biological function of LINC01354 in osteosarcoma. METHODS: Expression of LINC01354 in OS tissues, serum and cell lines was measured and the association between LINC01354 expression and clinicopathological factors was analyzed. The functional effects of LINC01354 were examined in vitro by using transwell assays, western blot, immunohistochemistry (IHC) and in vivo in a xenograft tumor mouse model. RESULTS: LINC01354 was overexpressed in OS tissues, serum and cells. LINC01354 overexpression promoted OS cells invasion, EMT and integrin ß1 expression, while knockdown of LINC01354 inhibited OS cell invasion, epithelial-mesenchymal transition (EMT) and integrin ß1 expression. In addition, integrin-ß1 blockage with MAB13 antibody abrogated the effects of LINC01354 overexpression on promoting OS cells invasion and EMT. In addition, LINC01354 promoted OS cell metastasis in vivo. CONCLUSION: LINC01354 promote OS cell EMT and invasion through up-regulating integrin ß1. Our study suggested that LINC01354 may be regarded as a potential target for the clinical treatment of OS.


Assuntos
Integrina beta1/metabolismo , Osteossarcoma/tratamento farmacológico , RNA Longo não Codificante/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Camundongos , Regulação para Cima
14.
Proc Natl Acad Sci U S A ; 117(12): 6686-6696, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32161126

RESUMO

Cytotoxic CD8+ T cells can effectively kill target cells by producing cytokines, chemokines, and granzymes. Expression of these effector molecules is however highly divergent, and tools that identify and preselect CD8+ T cells with a cytotoxic expression profile are lacking. Human CD8+ T cells can be divided into IFN-γ- and IL-2-producing cells. Unbiased transcriptomics and proteomics analysis on cytokine-producing fixed CD8+ T cells revealed that IL-2+ cells produce helper cytokines, and that IFN-γ+ cells produce cytotoxic molecules. IFN-γ+ T cells expressed the surface marker CD29 already prior to stimulation. CD29 also marked T cells with cytotoxic gene expression from different tissues in single-cell RNA-sequencing data. Notably, CD29+ T cells maintained the cytotoxic phenotype during cell culture, suggesting a stable phenotype. Preselecting CD29-expressing MART1 TCR-engineered T cells potentiated the killing of target cells. We therefore propose that CD29 expression can help evaluate and select for potent therapeutic T cell products.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/imunologia , Integrina beta1/metabolismo , Interferon gama/metabolismo , Interleucina-2/metabolismo , Melanoma/patologia , Linfócitos T Citotóxicos/imunologia , Perfilação da Expressão Gênica , Humanos , Melanoma/imunologia , Melanoma/metabolismo , Prognóstico , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Taxa de Sobrevida
15.
Nat Commun ; 11(1): 1211, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139701

RESUMO

Tumor metastasis is a hallmark of cancer. Metastatic cancer cells often reside in distal tissues and organs in their dormant state. Mechanisms underlying the pre-metastatic niche formation are poorly understood. Here we show that in a colorectal cancer (CRC) model, primary tumors release integrin beta-like 1 (ITGBL1)-rich extracellular vesicles (EVs) to the circulation to activate resident fibroblasts in remote organs. The activated fibroblasts induce the pre-metastatic niche formation and promote metastatic cancer growth by secreting pro-inflammatory cytokine, such as IL-6 and IL-8. Mechanistically, the primary CRC-derived ITGBL1-enriched EVs stimulate the TNFAIP3-mediated NF-κB signaling pathway to activate fibroblasts. Consequently, the activated fibroblasts produce high levels of pro-inflammatory cytokines to promote metastatic cancer growth. These findings uncover a tumor-stromal interaction in the metastatic tumor microenvironment and an intimate signaling communication between primary tumors and metastases through the ITGBL1-loaded EVs. Targeting the EVs-ITGBL1-CAFs-TNFAIP3-NF-κB signaling axis provides an attractive approach for treating metastatic diseases.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Vesículas Extracelulares/metabolismo , Fibroblastos/patologia , Integrina beta1/metabolismo , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Luciferases/metabolismo , Camundongos , NF-kappa B/metabolismo , Metástase Neoplásica , Transdução de Sinais , Distribuição Tecidual , Transcrição Genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo
16.
Biochim Biophys Acta Rev Cancer ; 1873(2): 188355, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32135169

RESUMO

The human ether-à-go-go related gene (HERG) encodes the alpha subunit of Kv11.1, which is a voltage-gated K+ channel protein mainly expressed in heart and brain tissue. HERG plays critical role in cardiac repolarization, and mutations in HERG can cause long QT syndrome. More recently, evidence has emerged that HERG channels are aberrantly expressed in many kinds of cancer cells and play important roles in cancer progression. HERG could therefore be a potential biomarker for cancer and a possible molecular target for anticancer drug design. HERG affects a number of cellular processes, including cell proliferation, apoptosis, angiogenesis and migration, any of which could be affected by dysregulation of HERG. This review provides an overview of available information on HERG channel as it relates to cancer, with focus on the mechanism by which HERG influences cancer progression. Molecular docking attempts suggest two possible protein-protein interactions of HERG with the ß1-integrin receptor and the transcription factor STAT-1 as novel HERG-directed therapeutic targeting which avoids possible cardiotoxicity. The role of epigenetics in regulating HERG channel expression and activity in cancer will also be discussed. Finally, given its inherent extracellular accessibility as an ion channel, we discuss regulatory roles of this molecule in cancer physiology and therapeutic potential. Future research should be directed to explore the possibilities of therapeutic interventions targeting HERG channels while minding possible complications.


Assuntos
Carcinogênese/patologia , Canal de Potássio ERG1/metabolismo , Integrina beta1/metabolismo , Neoplasias/patologia , Fator de Transcrição STAT1/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Carcinogênese/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/química , Canal de Potássio ERG1/genética , Epigênese Genética/efeitos dos fármacos , Fluoxetina/farmacologia , Fluoxetina/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Síndrome do QT Longo/genética , Potenciais da Membrana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Mutação , Miócitos Cardíacos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Conformação Proteica em alfa-Hélice , Mapeamento de Interação de Proteínas , Estrutura Quaternária de Proteína , Piridinas/farmacologia , Piridinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Sulfanilamidas/farmacologia , Sulfanilamidas/uso terapêutico
17.
Cancer Invest ; 38(4): 228-239, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32208057

RESUMO

The aim of this study was to characterize both by flow cytometry analysis and immunohistochemistry cervix uteri cells of nulliparous women screened for cervical intraepithelial neoplasia (CIN) in comparison to a group without CIN by using mesenchymal stem cell-like and hematopoietic lineage markers. A significant expression for CD29, CD38, HLA-I, and HLA-II was correlated positively to the CIN degree and it was more relevant in patients positive for human papilloma virus (HPV). Thus, identification and detailed characterization of pluripotent resident in uteri cells could be a promising therapeutic target.


Assuntos
Neoplasia Intraepitelial Cervical/patologia , Colo do Útero/citologia , Células-Tronco Neoplásicas/patologia , Infecções por Papillomavirus/patologia , Neoplasias do Colo do Útero/patologia , ADP-Ribosil Ciclase 1/análise , ADP-Ribosil Ciclase 1/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Adulto , Biópsia , Neoplasia Intraepitelial Cervical/imunologia , Neoplasia Intraepitelial Cervical/virologia , Colo do Útero/imunologia , Colo do Útero/patologia , Colo do Útero/virologia , Inibidor p16 de Quinase Dependente de Ciclina/análise , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imuno-Histoquímica , Imunofenotipagem , Integrina beta1/análise , Integrina beta1/imunologia , Integrina beta1/metabolismo , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Gradação de Tumores , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/virologia , Papillomaviridae/imunologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Estudos Prospectivos , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologia
18.
Zool Res ; 41(2): 203-207, 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32150793

RESUMO

Gap junctions regulate intercellular communication between Sertoli cells and germ cells in male testes and play vital functions in spermatogenesis. Many factors in animal breeding and husbandry can induce oxidative stress, which can impair the testis microenvironment and male animal fertility. However, the underlying mechanisms are largely unknown. Recently, we identified that androgen signals promote the expression of connexin-43 (Cx43), a key component of gap junctions, to regulate spermatogenesis. Thus, we asked whether hyperactive reactive oxygen species (ROS) can impair gap junctions by interfering with Cx43 in porcine testes. Using a porcine Sertoli cell in vitro system, we found that hyperactive ROS caused extensive apoptosis in Sertoli cells, remarkable decrease in Cx43 expression, and failed maintenance of co-cultured spermatogonial stem cells (SSCs), indicating that ROS impaired the function of Sertoli cells and promoted loss of SSCs. This observation provides a possible mechanism for the impact of ROS on fertility of male animals.


Assuntos
Conexina 43/metabolismo , Integrina beta1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células de Sertoli/metabolismo , Suínos/metabolismo , Animais , Masculino
19.
Infect Immun ; 88(5)2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32094259

RESUMO

The intracellular lifestyle of bacteria is widely acknowledged to be an important mechanism in chronic and recurring infection. Among the Staphylococcus genus, only Staphylococcus aureus and Staphylococcus pseudintermedius have been clearly identified as intracellular in nonprofessional phagocytic cells (NPPCs), for which the mechanism is mainly fibronectin-binding dependent. Here, we used bioinformatics tools to search for possible new fibronectin-binding proteins (FnBP-like) in other Staphylococcus species. We found a protein in Staphylococcus delphini called Staphylococcus delphini surface protein Y (SdsY). This protein shares 68% identity with the Staphylococcus pseudintermedius surface protein D (SpsD), 36% identity with S. aureus FnBPA, and 39% identity with S. aureus FnBPB. The SdsY protein possesses the typical structure of FnBP-like proteins, including an N-terminal signal sequence, an A domain, a characteristic repeated pattern, and an LPXTG cell wall anchor motif. The level of adhesion to immobilized fibronectin was significantly higher in all S. delphini strains tested than in the fibronectin-binding-deficient S. aureus DU5883 strain. By using a model of human osteoblast infection, the level of internalization of all strains tested was significantly higher than with the invasive-incompetent S. aureus DU5883. These findings were confirmed by phenotype restoration after transformation of DU5883 by a plasmid expression vector encoding the SdsY repeats. Additionally, using fibronectin-depleted serum and murine osteoblast cell lines deficient for the ß1 integrin, the involvement of fibronectin and ß1 integrin was demonstrated in S. delphini internalization. The present study demonstrates that additional staphylococcal species are able to invade NPPCs and proposes a method to identify FnBP-like proteins.


Assuntos
Fagócitos/metabolismo , Fagócitos/microbiologia , Staphylococcus/metabolismo , Staphylococcus/patogenicidade , Adesinas Bacterianas/metabolismo , Animais , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Parede Celular/metabolismo , Fibronectinas/metabolismo , Humanos , Integrina beta1/metabolismo , Camundongos
20.
Reprod Sci ; 27(1): 132-144, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32046405

RESUMO

This study aimed to investigate the regulatory mechanism of circular RNA CSPP1 (hsa_circ_CSPP1) in cervical cancer. Based on GEO database, differentially expressed circRNAs and mRNAs related to cervical cancer were screened out by R software. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) term analysis were performed to analyze the functional and pathway enrichment of identified DEGs. In addition, Cytoscape software was used to build interaction network of DEGs. The mRNA expressions were examined by qRT-PCR. Western blot was conducted to view the expression of proteins. Cell proliferation and apoptosis were respectively evaluated using CCK-8 assay and flow cytometry, whereas cell migration abilities were detected by Transwell assay. The relationship among factors was validated by dual-luciferase reporter gene assay. The influence in cervical tumor growth was further verified through nude mouse model in vivo. Hsa_circ_CSPP1 and ITGB1 were high-expressed in cervical cancer, while miR-361-5p was low-expressed. Hsa_circ_CSPP1 knockdown or miR-361-5p overexpression could suppress cervical cancer cell proliferation and migration, whereas promoted cell apoptosis. In addition, further experiments demonstrated that both hsa_circ_CSPP1 and ITGB1 mRNA were targets of miR-361-5p. Repressing hsa_circ_CSPP1 restrained cell viability and mobility and induced apoptosis through sponging miR-361-5p. Meanwhile, miR-361-5p also inhibited cervical cancer tumorigenesis via downregulation of ITGB1. Knockdown of hsa_circ_CSPP1 impeded tumor growth through suppressing the expression of downstream gene ITGB1, PI3K, and Akt. Circular RNA hsa_circ_CSPP1 regulates cell migration and proliferation in cervical cancer through miR-361-5p/ITGB1 in PI3K-Akt signaling pathway.


Assuntos
Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Integrina beta1/metabolismo , MicroRNAs/metabolismo , RNA Circular/metabolismo , Transdução de Sinais/fisiologia , Neoplasias do Colo do Útero/metabolismo , Apoptose/fisiologia , Linhagem Celular Tumoral , Biologia Computacional , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias do Colo do Útero/patologia
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