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1.
Anticancer Res ; 39(10): 5311-5327, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570425

RESUMO

BACKGROUND/AIM: MiR-221, often described both as an oncogenic microRNA and as a tumour suppressor, targets mRNAs involved in carcinogenesis. While other oncogenic microRNAs showed correlations with prostate cancer cell lines' aggressiveness, miR-221 showed an unusual overexpression in PC3. MATERIALS AND METHODS: CRISPR was used to delete miR-221 from PC3 cells. Analysing the characteristics of PC3miR-221del cells, a reduced growth rate and expression of cell-cycle genes was observed. In global gene expression/ontology analysis of PC3miR-221del cells, cell-cell and cell-substrate adhesion pathways were found to be greatly affected. In addition, reduced levels of adhesion, invasion and motility for PC3miR-221del cells, a change in F-actin localisation and a reduction of EMT markers were observed. RESULTS: The tumour suppressor gene, DIRAS3, was a predicted target of miR-221. In PC3miR-221del cells DIRAS3 was up-regulated at the gene and protein level. Ectopic expression of DIRAS3 in PC3wt cells recapitulated the cellular morphology changes seen in PC3miR-221del cells. DIRAS3 3'UTR was more stable in PC3miR-221del cells, as measured by semi-quantitative PCR and luciferase fusion reporter assays. CONCLUSION: MiR-221 promotes aggressiveness of PC3 cells by down-regulating DIRAS3, and promoting epithelial-to-mesenchymal transition.


Assuntos
Adesão Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , MicroRNAs/genética , Deleção de Sequência/genética , Regiões 3' não Traduzidas/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Oncogenes/genética , Células PC-3 , Neoplasias da Próstata/genética , Regulação para Cima/genética , Proteínas rho de Ligação ao GTP/genética
2.
Cytogenet Genome Res ; 158(4): 205-212, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31434093

RESUMO

EHMT2 (euchromatic histone lysine methyltransferase 2), a histone methyltransferase, has been shown to be involved in multiple human cancers. In this study, we determined mRNA and protein expression of EHMT2 in cervical cancer cells and normal cervical epithelial cells. EHMT2 was inhibited with short hairpin RNA (shEHMT2) in cervical cancer cells. Cell viability, colony proliferation, apoptosis, adhesion, and invasion assays and Western blot were performed to assess the function of EHMT2. As a result, EHMT2 was upregulated in human cervical cancer cells compared to normal cervical epithelial cells. Suppression of EHMT2 expression impairs cell proliferation and induces apoptosis. Furthermore, EHMT2 silencing inhibited cell adhesion and invasion. Finally, knockdown of EHMT2 resulted in a reduction of the expression of the tumorigenic proteins Bcl-2, Mcl-1, and Survivin and in an increase in the expression of the anti-malignant protein E-cadherin. In conclusion, our data suggest that EHMT2 plays a key role in cell proliferation and metastatic capacity in cervical cancer cells and could serve as a potential therapeutic target.


Assuntos
Inativação Gênica , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/prevenção & controle , Neoplasias do Colo do Útero/enzimologia , Neoplasias do Colo do Útero/patologia , Apoptose/genética , Caderinas/biossíntese , Adesão Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Neoplasias do Colo do Útero/genética
3.
Int J Nanomedicine ; 14: 4133-4144, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31239672

RESUMO

Background: Although titanium dioxide nanotubes (TNTs) had great potential to promote osteogenesis, their weak bonding strength with titanium substrates greatly limited their clinical application. Purpose: The objective of this study was to maintain porosity and improve the stability of TNT coatings by preparing some micro-patterned mesoporous/nanotube (MP/TNT) structures via a photolithography-assisted anodization technology. Methods: The adhesion strength of different coatings was studied by ultrasonic cleaning machine and scratch tester. The early adhesion, spreading, proliferation and differentiation of MC3T3-E1 cells on different substrates were investigated in vitro by fluorescent staining, CCK8, alkaline phosphatase activity, mineralization and polymerase chain reaction assays, respectively. Results: Results of ultrasonic and scratch assays showed that the stability of TNTs (especially 125 nm) was significantly improved after being patterned with MP structures. In vitro cell assays further demonstrated that the insertion of MP structure into 125 nm TNT coating, which was denoted as MP125, could effectively improve the early adhesion, spreading and proliferation of surface MC3T3-E1 cells without damaging their osteogenic differentiation. Conclusion: We determined that the MP/TNT patterned samples (especially MP125) have excellent stability and osteogenesis properties, and may have better clinical application prospects.


Assuntos
Nanotubos/química , Osteogênese , Titânio/química , Adsorção , Fosfatase Alcalina/metabolismo , Animais , Adesão Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Forma Celular/genética , Sobrevivência Celular/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fluorescência , Regulação da Expressão Gênica , Humanos , Camundongos , Minerais/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Osteopontina/genética , Osteopontina/metabolismo , Porosidade , Água/química
4.
DNA Cell Biol ; 38(6): 549-560, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31120353

RESUMO

Proper course of folliculogenesis and oogenesis have an enormous impact on female fertility. Both processes take place in the ovary and involve not only the maturing germ cell, but also few types of somatic cells that assist the ovarian processes and mediate the dialog with the oocyte. These cells, granulosa and theca, are heavily involved in essential reproductive processes, such as ovulation, fertilization, and embryo implantation. In this study, we have used the expressive microarray approach to analyze the transcriptome of porcine granulosa cells, during short-term in vitro culture. We have further selected differentially expressed gene ontologies, involved in cell proliferation, migration, adhesion, and tissue development, namely, "cell-cell adhesion," "cell motility," "cell proliferation," "tissue development," and "tissue migration" to screen them for the possibility of discovery of new markers of those processes. A total of 303 genes, expression of which varied significantly in different culture periods and belonged to the analyzed ontology groups, were detected, of which 15 that varied the most (between 0 and 48 h of culture) were selected for validation. As the validation confirmed the transcriptomic patterns, 10 genes of biggest changes in expression (CAV1, IGFBP5, ITGB3, FN1, ITGA2, LAMB1, POSTN, FAM83D, KIF14, and CDK1) were analyzed, described, and referred to the context of the study, with the most promising new markers and further proof for the viability of the currently recognized ones detailed. Overall, the study provided valuable insight into the molecular functioning of in vitro granulosa cell cultures.


Assuntos
Adesão Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , Células da Granulosa/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Células da Granulosa/citologia , Células da Granulosa/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Suínos
5.
Biomed Res Int ; 2019: 3638469, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058187

RESUMO

Eosinophil asthma is characterized by the infiltration of eosinophils to the bronchial epithelium. The toxic cationic protein released by eosinophils, mainly major basic protein (MBP), is one of the most important causative factors of epithelium damage. Poly-L-Arginine (PLA) is a kind of synthetic cationic polypeptides, which is widely used to mimic the effects of MBP on epithelial cells in vitro. However, little is known about the changes of differentially expressed genes (DEGs) and transcriptome profiles in cationic protein stimulated epithelial cells. In this study, we compared the expression of DEGs and transcriptome profiles between PLA-treated airway epithelial cells NCI-H292 and control. The results showed that there were a total of 230 DEGs, of which 86 were upregulated and 144 were downregulated. These DEGs were further analyzed using gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The results showed that the upregulated DEGs were involved in cholesterol synthesis, protein binding, and composition of cellular membranes, mainly enriched in metabolic and biosynthesis pathways. While downregulated DEGs were implicated in cell adhesion, extracellular matrix (ECM) composition and cytoskeleton and were enriched in ECM pathway. In conclusion, our research provided the mechanism of the cationic polypeptides acting on the airway epithelial cells on the basis of transcriptomic profile, and this could be regarded as important indications in unveiling the pathologic role of natural cationic proteins in the damage to epithelial cells of asthmatics.


Assuntos
Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Pulmão/metabolismo , Transcriptoma/genética , Cátions/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Colesterol/genética , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Redes Reguladoras de Genes/genética , Humanos , Pulmão/efeitos dos fármacos , Peptídeos/farmacologia , Sequenciamento Completo do Exoma
6.
Lipids Health Dis ; 18(1): 98, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975221

RESUMO

BACKGROUND: Essential hypertension can cause many kinds of cardiovascular diseases. The pathogenesis of essential hypertension is very complex, and the mechanism is still unclear. The microRNAs have been identified as novel biomarkers for pre-diagnosis and prognosis of hypertension. However, the kinds of microRNAs that can be used as specific biomarkers for hypertension are unknown. METHODS AND RESULTS: Plasma samples were isolated from Uyghur subjects with essential hypertension and the healthy individuals. Microarray was used to identify differentially expressed microRNAs. The microarray data were clustered and annotated with online software. The target genes of differentially expressed microRNAs were also analyzed. The microarray results were further verified by quantitative real-time PCR. We identified 257 microRNAs that were differentially expressed between patients with essential hypertension and the healthy individuals. These microRNAs had a total of 6580 target genes. The 47 microRNAs that had target genes, including 24 up-regulated and 23 down-regulated microRNAs, were further screened out to construct a reference set of potential microRNA biomarkers. Most of the 47 microRNAs were located at chromosome 19 (40 microRNAs) and chromosome 1 (45 microRNAs). Their target genes were mainly enriched in metal ion binding, transcription regulation, cell adhesion and junction, indicating that these candidate microRNAs may regulate mineral ion binding and cell communication process of essential hypertension. The quantitative real-time PCR results of miR-198 and miR-1183 (which were the two most significantly up-regulated microRNAs by microarray), and, miR-30e-5p and miR-144-3p (which were the two most significantly down-regulated microRNAs by microarray) were consistent with the microarray results. CONCLUSIONS: A reference set of potential microRNA biomarkers that may be involved in essential hypertension is constructed. Our study may provide experimental evidence for further studying the mechanism of essential hypertension.


Assuntos
Hipertensão Essencial/diagnóstico , Regulação da Expressão Gênica , MicroRNAs/genética , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Adesão Celular/genética , China , Mapeamento Cromossômico , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 19 , Diagnóstico Precoce , Hipertensão Essencial/sangue , Hipertensão Essencial/etnologia , Hipertensão Essencial/genética , Grupos Étnicos , Feminino , Humanos , Transporte de Íons/genética , Masculino , MicroRNAs/sangue , Análise em Microsséries , Anotação de Sequência Molecular , Prognóstico , Transdução de Sinais
7.
Int J Mol Sci ; 20(8)2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31027181

RESUMO

DUSP6/MKP3 is a dual-specific phosphatase that regulates extracellular regulated kinase ERK1/2 and ERK5 activity, with an increasingly recognized role as tumor suppressor. In silico studies from Gene expression Omnibus (GEO) and Cancer Genome atlas (TCGA) databases reveal poor prognosis in those Non-small cell lung cancer (NSCLC) patients with low expression levels of DUSP6. In agreement with these data, here we show that DUSP6 plays a major role in the regulation of cell migration, motility and tumor growth. We have found upregulation in the expression of several genes involved in epithelial to mesenchymal transition (EMT) in NSCLC-DUSP6 depleted cells. Data obtained in RNA-seq studies carried out in DUSP6 depleted cells identified EGFR, TGF-ß and WNT signaling pathways and several genes such as VAV3, RUNXR2, LEF1, FGFR2 whose expression is upregulated in these cells and therefore affecting cellular functions such as integrin mediated cell adhesion, focal adhesion and motility. Furthermore, EGF signaling pathway is activated via ERK5 and not ERK1/2 and TGF-ß via SMAD2/3 in DUSP6 depleted cells. In summary DUSP6 is a tumor suppressor in NSCLC and re-establishment of its expression may be a potential strategy to revert poor outcome in NSCLC patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Fosfatase 6 de Especificidade Dupla/genética , Genes Supressores de Tumor , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Citoesqueleto de Actina/metabolismo , Adenocarcinoma de Pulmão/enzimologia , Adenocarcinoma de Pulmão/patologia , Junções Aderentes/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Forma Celular/genética , Progressão da Doença , Fosfatase 6 de Especificidade Dupla/metabolismo , Adesões Focais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Camundongos Nus , Fator de Crescimento Transformador beta/metabolismo
8.
PLoS Genet ; 15(3): e1007967, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30901340

RESUMO

Mast cell tumours are the most common type of skin cancer in dogs, representing a significant concern in canine health. The molecular pathogenesis is largely unknown, but breed-predisposition for mast cell tumour development suggests the involvement of inherited genetic risk factors in some breeds. In this study, we aimed to identify germline risk factors associated with the development of mast cell tumours in Labrador Retrievers, a breed with an elevated risk of mast cell tumour development. Using a methodological approach that combined a genome-wide association study, targeted next generation sequencing, and TaqMan genotyping, we identified a synonymous variant in the DSCAM gene on canine chromosome 31 that is associated with mast cell tumours in Labrador Retrievers. DSCAM encodes a cell-adhesion molecule. We showed that the variant has no effect on the DSCAM mRNA level but is associated with a significant reduction in the level of the DSCAM protein, suggesting that the variant affects the dynamics of DSCAM mRNA translation. Furthermore, we showed that the variant is also associated with mast cell tumours in Golden Retrievers, a breed that is closely related to Labrador Retrievers and that also has a predilection for mast cell tumour development. The variant is common in both Labradors and Golden Retrievers and consequently is likely to be a significant genetic contributor to the increased susceptibility of both breeds to develop mast cell tumours. The results presented here not only represent an important contribution to the understanding of mast cell tumour development in dogs, as they highlight the role of cell adhesion in mast cell tumour tumourigenesis, but they also emphasise the potential importance of the effects of synonymous variants in complex diseases such as cancer.


Assuntos
Moléculas de Adesão Celular/genética , Mastocitoma Cutâneo/genética , Mastocitoma Cutâneo/veterinária , Animais , Adesão Celular/genética , Doenças do Cão/genética , Cães , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Células Germinativas , Mutação em Linhagem Germinativa/genética , Mastócitos/metabolismo , Mastócitos/fisiologia , Mastocitoma Cutâneo/metabolismo , Mastocitose Cutânea/genética , Fatores de Risco , Mutação Silenciosa/genética , Neoplasias Cutâneas/genética
9.
Nat Commun ; 10(1): 1398, 2019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30923315

RESUMO

The roles of Plant Homeodomain (PHD) fingers in catalysis of histone modifications are unknown. We demonstrated that the PHD finger of Ubiquitin Protein Ligase E3 Component N-Recognin7 (UBR7) harbors E3 ubiquitin ligase activity toward monoubiquitination of histone H2B at lysine120 (H2BK120Ub). Purified PHD finger or full-length UBR7 monoubiquitinated H2BK120 in vitro, and loss of UBR7 drastically reduced H2BK120Ub genome-wide binding sites in MCF10A cells. Low UBR7 expression was correlated with occurrence of triple-negative breast cancer and metastatic tumors. Consistently, UBR7 knockdown enhanced the invasiveness, induced epithelial-to-mesenchymal transition and promoted metastasis. Conversely, ectopic expression of UBR7 restored these cellular phenotypes and reduced tumor growth. Mechanistically, UBR7 loss reduced H2BK120Ub levels on cell adhesion genes, including CDH4, and upregulated the Wnt/ß-Catenin signaling pathway. CDH4 overexpression could partially revert UBR7-dependent cellular phenotypes. Collectively, our results established UBR7 as a histone H2B monoubiquitin ligase that suppresses tumorigenesis and metastasis of triple-negative breast cancer.


Assuntos
Carcinogênese/genética , Código das Histonas/genética , Histonas/metabolismo , Dedos de Zinco PHD/genética , Neoplasias de Mama Triplo Negativas/genética , Ubiquitina-Proteína Ligases/genética , Animais , Caderinas/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Transplante Heterólogo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Via de Sinalização Wnt
10.
Int J Mol Med ; 43(4): 1817-1829, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816445

RESUMO

Previous studies have demonstrated that the dysregulation of microRNAs (miRs) is frequently associated with cancer progression. Deregulation of miR­106b­3p has been observed in various types of human cancer. However, the biological function of miR­106b­3p in esophageal squamous cell carcinoma (ESCC) remains unclear. Thus, the aim of this study was to investigate the role of miR­106b­3p in ESCC. In the current study, the results indicated that miR­106b­3p was upregulated in ESCC cell lines and tissues. An increase in miR­106b­3p using miR mimics significantly promoted the proliferation of ESCC cells in vitro. Furthermore, the results demonstrated that miR­106b­3p overexpression promoted migration, invasion and epithelial­mesenchymal transition (EMT) of ESCC cells. In addition, zinc and ring finger 3 (ZNRF3) was identified as a target of miR­106b­3p in ESCC cells, and the ZNRF3 expression level was inversely associated with miR­106b­3p. It was also demonstrated that miR­106b­3p has a role in EMT by regulating Wnt/ß­catenin signaling pathway in ESCC. In conclusion, these data suggested that miR­106b­3p promotes cell proliferation and invasion, partially by downregulating ZNRF3 and inducing EMT via Wnt/ß­catenin signaling in ESCC cells. Thus, miR­106b­3p and ZNRF3 may be novel molecular targets for the future treatment of ESCC.


Assuntos
Transição Epitelial-Mesenquimal/genética , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , MicroRNAs/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Bases , Adesão Celular/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Ubiquitina-Proteína Ligases/genética , Regulação para Cima/genética , Via de Sinalização Wnt/genética
11.
Int J Mol Sci ; 20(6)2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30884801

RESUMO

The small GTPase Rho and its downstream effector, Rho-kinase (ROCK), regulate various cellular functions, including organization of the actin cytoskeleton, cell adhesion and migration. A pro-inflammatory lipid mediator, lysophosphatidic acid (LPA), is a potent activator of the Rho/ROCK signalling pathway and has been shown to induce the expression of chemokines and cell adhesion molecules (CAMs). In the present study, we aimed to elucidate the precise mechanism by which ROCK regulates LPA-induced expressions and functions of chemokines and CAMs. We observed that ROCK blockade reduced LPA-induced phosphorylation of IκBα and inhibited NF-κB RelA/p65 phosphorylation, leading to attenuation of RelA/p65 nuclear translocation. Furthermore, small interfering RNA-mediated ROCK isoform knockdown experiments revealed that LPA induces the expression of monocyte chemoattractant protein-1 (MCP-1) and E-selectin via ROCK2 in human aortic endothelial cells (HAECs). Importantly, we found that ROCK2 but not ROCK1 controls LPA-induced monocytic migration and monocyte adhesion toward endothelial cells. These findings demonstrate that ROCK2 is a key regulator of endothelial inflammation. We conclude that targeting endothelial ROCK2 is potentially effective in attenuation of atherosclerosis.


Assuntos
Aterosclerose/genética , Células Endoteliais/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Quinases Associadas a rho/genética , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Aorta/citologia , Aorta/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Adesão Celular/genética , Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/genética , Selectina E/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Quinase I-kappa B/genética , Monócitos/efeitos dos fármacos , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/genética , Quinases Associadas a rho/metabolismo
12.
Mater Sci Eng C Mater Biol Appl ; 99: 129-139, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30889664

RESUMO

The aim of this study is to evaluate a core-shell nanofiber as a useful matrix for tuning Rosuvastatin (RSV) release and osteogenic differentiation in vitro. Polyvinyl alcohol (PVA) and silk fibroin were used as the shell and the core, respectively. To obtain a linear and beadless core-shell structure and an optimal release profile, the shell/core flow rate ratio was varied (0, 0.4, 0.6, 0.8, and 1). Formation of continuous nanofibers with an obvious core/sheath structure was proved using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Differential scanning calorimetry indicated the presence of two distinct phase structures in the nanofibers. Also, RSV molecules were dispersed in an amorphous state in the nanofibers. The in vitro release profile of the core-shell structure exhibited a biphasic release profile and the amount of released RSV was controlled by adjusting the shell flow rate. Human adipose-derived stem cells cultured on the RSV loaded nanofibers were found to improve cell proliferation and assist osteogenic differentiation as revealed by Alizarin red staining and real-time RT-PCR.


Assuntos
Tecido Adiposo/citologia , Fibroínas/química , Nanofibras/química , Osteogênese/efeitos dos fármacos , Álcool de Polivinil/química , Rosuvastatina Cálcica/farmacologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Adulto , Animais , Bombyx , Varredura Diferencial de Calorimetria , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Liberação Controlada de Fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Pessoa de Meia-Idade , Nanofibras/ultraestrutura , Osteogênese/genética , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Molhabilidade
13.
Gynecol Obstet Invest ; 84(4): 396-406, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30759440

RESUMO

BACKGROUND/AIMS: The study aimed to evaluate molecular changes related to trophoblast adhesion in placenta accreta spectrum (PAS) disorders. METHODS: A retrospective analysis of 10 PAS cases in which both the trophoblast adherent site and the non-adherent site were identified was performed in April 2010 and March 2013. Microarray analysis and reverse transcription polymerase chain reaction (RT-PCR) analyses were performed to extract upregulated genes in the adherent site. Gene expression changes were examined by immunohistochemistry. RESULTS: Microarray analysis showed that 157 transcripts were > 3-fold upregulated, including the following: a disintegrin and metalloproteinase-28 (ADAM28), 3.10-fold; cathepsin V (CTSV), 3.73-fold; cathepsin S (CTSS), 3.46-fold; and matrix metalloproteinase-19 (MMP19), 3.41-fold. RT-PCR showed relatively high mRNA expressions. On immunohistochemistry, extravillous trophoblast (EVT) at the non-adherent site showed weak or no CTSV expression, whereas EVT that invaded myometrium at the adherent site showed strong expression (histological score, median [min-max], 115.6 [37.6-153.6] vs. 184.8 [56.4-222.8], p < 0.05). MMP19 showed moderate staining, with no difference between the adherent and non-adherent sites. ADAM28 and CTSS showed weak or no staining. DISCUSSION: This limited study suggests that CTSV may be involved in the pathogenesis of PAS.


Assuntos
Catepsinas/metabolismo , Adesão Celular/genética , Cisteína Endopeptidases/metabolismo , Placenta Acreta/genética , Trofoblastos/metabolismo , Proteínas ADAM/metabolismo , Adulto , Feminino , Humanos , Imuno-Histoquímica , Metaloproteinases da Matriz Secretadas/metabolismo , Miométrio/metabolismo , Placenta/metabolismo , Gravidez , Estudos Retrospectivos
14.
Proc Natl Acad Sci U S A ; 116(8): 2837-2842, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718416

RESUMO

Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N'-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential.


Assuntos
Galectina 1/química , Galectina 3/química , Glicoconjugados/química , Polissacarídeos/química , Amino Açúcares/química , Amino Açúcares/metabolismo , Sítios de Ligação , Adesão Celular/genética , Proliferação de Células/genética , Galectina 1/genética , Galectina 3/genética , Humanos , Lactose/química , Ligantes , Nanopartículas/química , Polissacarídeos/genética
15.
Proc Natl Acad Sci U S A ; 116(9): 3536-3545, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808747

RESUMO

Collective cell migration is required for normal embryonic development and contributes to various biological processes, including wound healing and cancer cell invasion. The M-Ras GTPase and its effector, the Shoc2 scaffold, are proteins mutated in the developmental RASopathy Noonan syndrome, and, here, we report that activated M-Ras recruits Shoc2 to cell surface junctions where M-Ras/Shoc2 signaling contributes to the dynamic regulation of cell-cell junction turnover required for collective cell migration. MCF10A cells expressing the dominant-inhibitory M-RasS27N variant or those lacking Shoc2 exhibited reduced junction turnover and were unable to migrate effectively as a group. Through further depletion/reconstitution studies, we found that M-Ras/Shoc2 signaling contributes to junction turnover by modulating the E-cadherin/p120-catenin interaction and, in turn, the junctional expression of E-cadherin. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphoregulation of p120-catenin and required downstream ERK cascade activation. Strikingly, cells rescued with the Noonan-associated, myristoylated-Shoc2 mutant (Myr-Shoc2) displayed a gain-of-function (GOF) phenotype, with the cells exhibiting increased junction turnover and reduced E-cadherin/p120-catenin binding and migrating as a faster but less cohesive group. Consistent with these results, Noonan-associated C-Raf mutants that bypass the need for M-Ras/Shoc2 signaling exhibited a similar GOF phenotype when expressed in Shoc2-depleted MCF10A cells. Finally, expression of the Noonan-associated Myr-Shoc2 or C-Raf mutants, but not their WT counterparts, induced gastrulation defects indicative of aberrant cell migration in zebrafish embryos, further demonstrating the function of the M-Ras/Shoc2/ERK cascade signaling axis in the dynamic control of coordinated cell movement.


Assuntos
Adesão Celular/genética , Movimento Celular/genética , Desenvolvimento Embrionário/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Animais , Caderinas/genética , Mutação com Ganho de Função/genética , Gastrulação/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Síndrome de Noonan/genética , Síndrome de Noonan/fisiopatologia , Ligação Proteica , Peixe-Zebra/genética
16.
Int J Mol Sci ; 20(3)2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30736330

RESUMO

The bone marrow (BM) microenvironment plays an important role in supporting proliferation, survival and drug resistance of Multiple Myeloma (MM) cells. MM cells adhere to bone marrow stromal cells leading to the activation of tumour-promoting signaling pathways. Activation of the NFκB pathway, in particular, is central to the pathogenesis of MM. Tumour necrosis factor receptor-associated factor 6 (TRAF6) is a key mediator of NFκB activation and has previously been highlighted as a potential therapeutic target in MM. Here, we demonstrate that adherence of MM cell lines to stromal cells results in a reciprocal increase in TRAF6 expression. Knockdown of TRAF6 expression attenuates the ability of MM cells to bind to stromal cells and this is associated with a decrease in NFκB-induced expression of the adhesion molecules ICAM1 and VCAM1. Finally, we show that knockdown of TRAF6 sensitizes MM cells to treatment with bortezomib when co-cultured with stromal cells. Inhibiting TRAF6 represents a promising strategy to target MM cells in the BM microenvironment.


Assuntos
Inativação Gênica , Células-Tronco Mesenquimais/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Bortezomib/farmacologia , Adesão Celular/genética , Linhagem Celular Tumoral , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Mieloma Múltiplo/patologia , NF-kappa B/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos
17.
Biomed Res ; 40(1): 37-49, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30787262

RESUMO

The tumor suppressor gene TP53 (gene) codes for a transcription factor which transactivates its target genes responsible for cell cycle arrest, DNA repair, apoptosis, and senescence. TP53 is well known to be the most frequent target of genetic mutations in nearly half of human cancers including oral squamous cell carcinoma (OSCC). Many p53 mutants including R248Q and R248W not only lose its tumor-suppressor activities, but also interfere with the functions of wild-type p53; this is so-called dominant-negative (DN) mutation. The DN p53 mutation is a predictor of poor outcome in patients with various cancers, and also a risk factor for metastatic recurrence in patients with OSCC. Recently it has been reported that DN p53 mutants acquire new oncogenic activities, which is named gain-of-function (GOF). This study aimed at determining whether R248Q and R248W were involved in OSCC cells' acquiring aggressive phenotypes, using SAS, HSC4 and Ca9-22 cell lines. First, two mutants p53, R248Q and R248W, were respectively transfected into SAS cells harboring recessive-type p53 (E336X). As a result, SAS cells expressing R248Q showed highly spreading, motile and invasive activities compared to parent or mock-transfected cells whereas those expressing R248W did not increase those activities. Secondly, in HSC4 cells harboring R248Q and Ca9-22 cells harboring R248W, expressions of the mutants p53 were inhibited by the transfection with siRNAs targeting p53. The inhibition of the mutants p53 decreased spreading, motile and invasive activities of HSC4 cells whereas it did not affect those activities of Ca9-22 cells. These findings suggest that R248Q p53 mutation, but not R248W p53 mutation, induces more motile and invasive potentials in human OSCC cells.


Assuntos
Alelos , Substituição de Aminoácidos , Movimento Celular/genética , Genes Dominantes , Mutação , Proteína Supressora de Tumor p53/genética , Apoptose/genética , Carcinoma de Células Escamosas/genética , Adesão Celular/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Matriz Extracelular/metabolismo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Neoplasias Bucais/genética , Ligação Proteica , RNA Interferente Pequeno/genética , Elementos de Resposta , Proteína Supressora de Tumor p53/metabolismo
18.
Proc Natl Acad Sci U S A ; 116(9): 3524-3529, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30733287

RESUMO

RAP1-interacting adapter molecule (RIAM) mediates RAP1-induced integrin activation. The RAS-association (RA) segment of the RA-PH module of RIAM interacts with GTP-bound RAP1 and phosphoinositol 4,5 bisphosphate but this interaction is inhibited by the N-terminal segment of RIAM. Here we report the structural basis for the autoinhibition of RIAM by an intramolecular interaction between the IN region (aa 27-93) and the RA-PH module. We solved the crystal structure of IN-RA-PH to a resolution of 2.4-Å. The structure reveals that the IN segment associates with the RA segment and thereby suppresses RIAM:RAP1 association. This autoinhibitory configuration of RIAM can be released by phosphorylation at Tyr45 in the IN segment. Specific inhibitors of focal adhesion kinase (FAK) blocked phosphorylation of Tyr45, inhibited stimulated translocation of RIAM to the plasma membrane, and inhibited integrin-mediated cell adhesion in a Tyr45-dependent fashion. Our results reveal an unusual regulatory mechanism in small GTPase signaling by which the effector molecule is autoinhibited for GTPase interaction, and a modality of integrin activation at the level of RIAM through a FAK-mediated feedforward mechanism that involves reversal of autoinhibition by a tyrosine kinase associated with integrin signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Integrinas/química , Proteínas de Membrana/química , Conformação Proteica , Proteínas de Ligação a Telômeros/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Adesão Celular/genética , Membrana Celular/química , Cristalografia por Raios X , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/genética , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Humanos , Proteínas de Membrana/genética , Fosforilação , Transdução de Sinais , Proteínas de Ligação a Telômeros/genética
19.
Nano Lett ; 19(4): 2280-2290, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30775927

RESUMO

Cancer cell invasion through physical barriers in the extracellular matrix (ECM) requires a complex synergy of traction force against the ECM, mechanosensitive feedback, and subsequent cytoskeletal rearrangement. PDMS microchannels were used to investigate the transition from mesenchymal to amoeboid invasion in cancer cells. Migration was faster in narrow 3 µm-wide channels than in wider 10 µm channels, even in the absence of cell-binding ECM proteins. Cells permeating narrow channels exhibited blebbing and had smooth leading edge profiles, suggesting an ECM-induced transition from mesenchymal invasion to amoeboid invasion. Live cell labeling revealed a mechanosensing period in which the cell attempts mesenchymal-based migration, reorganizes its cytoskeleton, and proceeds using an amoeboid phenotype. Rho/ROCK (amoeboid) and Rac (mesenchymal) pathway inhibition revealed that amoeboid invasion through confined environments relies on both pathways in a time- and ECM-dependent manner. This demonstrates that cancer cells can dynamically modify their invasion programming to navigate physically confining matrix conditions.


Assuntos
Citoesqueleto/efeitos dos fármacos , Mesoderma/efeitos dos fármacos , Invasividade Neoplásica/genética , Neoplasias/genética , Fenômenos Biomecânicos , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Citoesqueleto/genética , Dimetilpolisiloxanos/química , Dimetilpolisiloxanos/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Humanos , Mesoderma/patologia , Invasividade Neoplásica/patologia , Neoplasias/patologia , Nylons/química , Nylons/farmacologia
20.
Proc Natl Acad Sci U S A ; 116(8): 3278-3287, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718399

RESUMO

Cells express a family of three inositol hexakisphosphate kinases (IP6Ks). Although sharing the same enzymatic activity, individual IP6Ks mediate different cellular processes. Here we report that IP6K3 is enriched at the leading edge of migrating cells where it associates with dynein intermediate chain 2 (DIC2). Using immunofluorescence microscopy and total internal reflection fluorescence microscopy, we found that DIC2 and IP6K3 are recruited interdependently to the leading edge of migrating cells, where they function coordinately to enhance the turnover of focal adhesions. Deletion of IP6K3 causes defects in cell motility and neuronal dendritic growth, eventually leading to brain malformations. Our results reveal a mechanism whereby IP6K3 functions in coordination with DIC2 in a confined intracellular microenvironment to promote focal adhesion turnover.


Assuntos
Dineínas do Citoplasma/genética , Dendritos/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Encéfalo/metabolismo , Encéfalo/patologia , Adesão Celular/genética , Movimento Celular/genética , Microambiente Celular/genética , Adesões Focais/genética , Células HEK293 , Humanos , Neurônios/metabolismo
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