RESUMEN
The adaptive immune response is reliant on a T cell's ability to migrate through blood, lymph, and tissue in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time under highly specific environmental conditions. These conditions may help elucidate mechanisms that regulate migration, improving our understanding of T cell kinetics and providing strong mechanistic evidence that is difficult to attain through animal experiments. A deeper understanding of the molecular interactions that impact cell migration is important to develop improved therapeutics.
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Linfocitos T CD8-positivos , Movimiento Celular , Animales , Ratones , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/citología , Movimiento Celular/fisiología , Movimiento Celular/inmunología , Ensayos de Migración Celular/métodosRESUMEN
The ability of cells to move and migrate is required during development, but also in the adult in processes such as wound healing and immune responses. In addition, cancer cells exploit the cells' ability to migrate and invade to spread into nearby tissue and eventually metastasize. The majority of cancer deaths are caused by metastasis and the process of cell migration is therefore intensively studied. A common way to study cell migration is to observe cells through an optical microscope and record their movements over time. However, segmenting and tracking moving cells in phase contrast time-lapse video sequences is a challenging task. Several tools to track the velocity of migrating cells have been developed. Unfortunately, most of the automated tools are made for fluorescence images even though unlabelled cells are often preferred to avoid phototoxicity. Consequently, researchers are constrained with laborious manual tracking tools using ImageJ or similar software. We have therefore developed a freely available, user-friendly, automated tracking tool called CellTraxx. This software makes it easy to measure the velocity and directness of migrating cells in phase contrast images. Here, we demonstrate that our tool efficiently recognizes and tracks unlabelled cells of different morphologies and sizes (HeLa, RPE1, MDA-MB-231, HT1080, U2OS, PC-3) in several types of cell migration assays (random migration, wound healing and cells embedded in collagen). We also provide a detailed protocol and download instructions for CellTraxx.
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Programas Informáticos , Cicatrización de Heridas , Adulto , Humanos , Movimiento Celular/fisiología , Células HeLa , Cicatrización de Heridas/fisiología , Ensayos de Migración Celular/métodos , Rastreo Celular/métodos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
SUMMARY: Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in various types of cancers including breast cancer. However, the role of AhR with its endogenous ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the progression of breast cancer remains poorly understood. We aimed to investigate cell proliferation and migration states in breast cancer after activating AhR with the endogenous ligand ITE. Breast cancer tissue was evaluated by cell lines, immunohistochemistry, reverse transcription-polymerase chain reaction, cell proliferation, flow cytometry, migration assays and western blot techniques. We found that AhR was widely expressed in breast cancer tissues and metastasis lymph node tissues, but not in normal tissues. The expression AhR was independent between the age, grades and TNM classifications for breast cancer tissues. ITE treatment significantly induced the activation of AhR in a time-dependent manner in both MCF-7 and T47D breast cancer cell lines. Meanwhile, ITE did not affect the cell migration but significantly suppressed the cell proliferation in estrogen receptor positive (ER+) MCF-7 andT47D cells, which probably attribute to the induction of cell cycle arrest in G1 phase and shortened S phase. Further mechanism study showed that ERK1/2 and AKT signaling were required for the activation of AhR in MCF-7 cells. These data suggest that AhR is a potential new target for treating patients with breast cancer. ITE may be more potentially used for therapeutic intervention for breast cancer with the kind of ER(+).
El receptor de hidrocarburo de arilo (AhR) es un factor de transcripción activado por ligando que se expresa en gran medida en varios tipos de cáncer, incluido el cáncer de mama. Sin embargo, el papel de AhR con su ligando endógeno 2- (1'H-indol-3'-carbonil)-tiazol-4-ácido carboxílico metil éster (ITE) en la progresión del cáncer de mama sigue siendo poco conocido. Nuestro objetivo fue investigar la proliferación celular y los estados de migración en el cáncer de mama después de activar AhR con el ligando endógeno ITE. El tejido de cáncer de mama se evaluó mediante líneas celulares, inmunohistoquímica, reacción en cadena de la polimerasa con transcriptasa inversa, proliferación celular, citometría de flujo, ensayos de migración y técnicas de transferencia Western. Descubrimos que AhR se expresó ampliamente en tejidos de cáncer de mama y en linfonodos con metástasis, pero no en tejidos normales. La expresión AhR fue independiente entre la edad, grados y clasificaciones TNM para tejidos de cáncer de mama. El tratamiento con ITE indujo significativamente la activación de AhR de manera dependiente del tiempo en las líneas celulares de cancer de mama MCF-7 y T47D. Mientras tanto, ITE no afectó la migración celular, pero suprimió significativamente la proliferación celular en células MCF-7 y T47D con receptor de estrógeno positivo (ER+), lo que probablemente se atribuye a la inducción de la detención del ciclo celular en la fase G1 y la fase S acortada. Un estudio adicional del mecanismo mostró que las señales de ERK1/2 y AKT eran necesarias para la activación de AhR en las células MCF-7. Estos datos sugieren que AhR es un nuevo objetivo potencial para el tratamiento de pacientes con cáncer de mama. ITE puede ser utilizado más potencialmente en la intervención terapéutica para el cáncer de mama con el tipo de ER (+).
Asunto(s)
Humanos , Femenino , Tiazoles/administración & dosificación , Neoplasias de la Mama/patología , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Indoles/administración & dosificación , Tiazoles/farmacología , Inmunohistoquímica , Receptores de Estrógenos , Western Blotting , Citocromo P-450 CYP1A1/genética , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayos de Migración Celular , Citocromo P-450 CYP1B1/genética , Citometría de Flujo , Indoles/farmacologíaRESUMEN
The enhanced availability of functional fibroblasts from precious tissue samples requires an ideal cell-culture system. Therefore, this study was designed to investigate the performance of caprine adult fibroblast cells (cadFibroblast) when cultivated in different culture media. The cadFibroblast cell lines from adult Barbari (Capra hircus) bucks were established and the effect of different media viz. DMEM/F-12 [with low-glucose (5.5 mM; DL) and high-glucose (30 mM; DH)], α-MEM [with low-glucose (5.5 mM; ML) and with high-glucose (30 mM; MH)], and fibroblast growth medium (FGM) were evaluated. Cells were then compared for growth characteristics and in-vitro dynamics through cellular morphology, proliferation, population-doubling time, double-immunocytochemistry, colony-forming units, wound healing, transwell migration, and differential expression of fibroblast-specific markers (FSP-1 and vimentin). The results of immunocytochemistry, transwell migration/invasion, and wound healing assays showed the superiority of DH over DL and other media tested. Whereas, similar effects of glucose supplementation and expression of FSP-1 were not observed in α-MEM. Transwell migration was significantly (p < 0.05) lower in FGM compared with other media tested. Overall, our results illustrate the media-dependent deviation in in-vitro dynamics and culture characteristics of cadFibroblasts that may be useful to develop strategies to cultivate these cells efficiently for research and downstream applications.
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Medios de Cultivo , Dermis , Fibroblastos , Cabras , Técnicas de Cultivo de Célula , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/microbiología , Medios de Cultivo/química , Medios de Cultivo/farmacología , Técnicas In Vitro , Dermis/citología , Animales , Línea Celular , Masculino , Glucosa/metabolismo , Perfilación de la Expresión Génica , Cicatrización de Heridas , Ensayos de Migración Celular , BiomarcadoresRESUMEN
Cancer is a cellular-based disease, so cytological diagnosis is one of the main challenges for its early detection. An extensive number of diagnostic methods have been developed to separate cancerous cells from normal ones, in electrical methods attract progressive attention. Identifying and specifying different cells requires understanding their dielectric and electric properties. This study evaluated MDA-MB-231, HUVEC, and MCF-10A cell lines, WBCs isolated from blood, and patient-derived cell samples with a cylindrical body with two transparent FTO (fluorine-doped tin oxide) plate electrodes. Cell mobility rates were recorded in response to these stimuli. It was observed that cancer cells demonstrate drastic changes in their motility in the presence and absence of an electric field (DC/AC). Also, solution viscosity's effect on cancer cells' capturing efficacy was evaluated. This research's main distinguished specification uses a non-microfluidic platform to detect and pathologically evaluate cytological samples with a simple, cheap, and repeatable platform. The capturing procedure was carried out on a cytological slide without any complicated electrode patterning with the ability of cytological staining. Moreover, this platform successfully designed and experimented with the invasion assay (the ability of captured cancer cells to invade normal cells).
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Electroforesis , Neoplasias , Electroforesis/instrumentación , Electroforesis/métodos , Neoplasias/diagnóstico , Neoplasias/patología , Invasividad Neoplásica , Electrodos , Humanos , Línea Celular Tumoral , Impresión Tridimensional , Separación Celular , Hipoxia de la Célula , Ensayos de Migración CelularRESUMEN
Migration assays are used to measure cell movement toward a variety of chemoattractants in a controlled environment. Here we describe a method for a Boyden chamber-based migration assay using conditioned media generated from the tumor, liver, and visceral adipose tissue of cancer patients.
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Factores Quimiotácticos , Quimiotaxis , Humanos , Movimiento Celular , Ensayos de Migración Celular/métodos , Línea Celular Tumoral , Medios de Cultivo CondicionadosRESUMEN
Cell migration and invasion have essential roles in both normal physiology and disease. As such, methodologies to assess cell migratory and invasive capacities are necessary to elucidate normal cell processes and underlying mechanisms of disease. Here, we describe commonly used transwell in vitro methods for the study of cell migration and invasion. The transwell migration assay involves the chemotaxis of cells through a porous membrane after the establishment of a chemoattractant gradient using two medium-filled compartments. The transwell invasion assay involves the addition of an extracellular matrix on top of the porous membrane which only permits chemotaxis of cells which possess invasive properties such as tumor cells.
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Quimiotaxis , Humanos , Movimiento Celular , Invasividad Neoplásica , Ensayos de Migración Celular , Línea Celular TumoralRESUMEN
The fundamental mechanism responsible for the aggressiveness of metastatic cancers such as triple-negative breast cancer (TNBC) is the epithelial-mesenchymal transition (EMT). In cancer microenvironments, the Phosphoinositide 3-kinases (PI3K)-Akt- mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in regulating the EMT mechanism. The current study focuses on the impacts of rapamycin, a newly retargeted chemotherapeutic agent against mTOR, and MicroRNA (miR)-122 on the aggressive behavior of TNBC. The half-maximal inhibitory concentration (IC50) of rapamycin on 4T1 cells was determined using an MTT assay. Also, miR-122 was transiently transfected into 4T1 cells to study its effect on the pathway. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess the expression level of central mTOR and EMT-related cascade genes. Moreover, cell mobility and migration were evaluated using scratch and migration assays, respectively. Both rapamycin and miR-122 significantly decreased the expression levels of PI3K, AKT, and mTOR, as well as ZeB1 and Snail genes. However, no significant change was observed in Twist gene expression. Furthermore, scratch and migration assays revealed that the migration of 4T1 cells was markedly reduced, especially following miR-122 induction. Our experimental findings and gene enrichment studies indicated that miR-122 mainly operates on multiple metabolic pathways, as well as EMT and mTOR, while rapamycin has restricted targets in cancer cells. Consequently, miR-122 can be considered a potential cancer microRNA therapy option, which can be validated in the future in animal studies to demonstrate its efficacy in cancer control.
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Transición Epitelial-Mesenquimal , MicroARNs , Transducción de Señal , Sirolimus , Animales , Ratones , Línea Celular Tumoral , Sirolimus/farmacología , MicroARNs/farmacología , Neoplasias de la Mama Triple Negativas , Transducción de Señal/efectos de los fármacos , Ensayos de Migración Celular , Transición Epitelial-Mesenquimal/efectos de los fármacos , Factores de Transcripción/metabolismo , Concentración 50 InhibidoraRESUMEN
Purpose: To investigate the role and mechanism of ß1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) in esophageal cancer (ESCA). Methods: The starBase database was used to evaluate the expression of B3GNT3. B3GNT3 function was measured using KYSE-30 and KYSE-410 cells of esophageal squamous cell carcinoma (ESCC) cell lines. The mRNA levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8, clone formation assay and transwell assay were used to detect the changes of proliferation, invasion and migration. Results: B3GNT3 expression was higher in ESCA tissues than in normal tissues. The overall survival rate of ESCA patients with high B3GNT3 expression was lower than that of ESCA patients with low B3GNT3 expression. In vitro functional experiments showed that the proliferation ability, migration and invasion ability of KYSE-30 and KYSE-410 cells with B3GNT3 interference were lower than those of the control, and the overexpression of B3GNT3 had the opposite effect. After silencing B3GNT3 expression in ESCC cell lines, the growth of both cell lines was inhibited and the invasiveness was decreased. Knockdown of B3GNT3 reduced the growth rate and Ki-67 expression level. Conclusion: B3GNT3, as an oncogene, may promote the growth, invasion and migration of ESCC cell.
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Oncogenes , N-Acetilglucosaminiltransferasas/análisis , Ensayos de Migración Celular , Transcriptoma , Carcinoma de Células Escamosas de Esófago , Neoplasias Esofágicas/fisiopatologíaRESUMEN
Microtubules, cylindrical assemblies of tubulin proteins with a 25 nm diameter and micrometer lengths, are a central part of the cytoskeleton and also serve as building blocks for nanobiodevices. Microtubule breaking can result from the activity of severing enzymes and mechanical stress. Breaking can lead to a loss of structural integrity, or an increase in the numbers of microtubules. We observed breaking of taxol-stabilized microtubules in a gliding motility assay where microtubules are propelled by surface-adhered kinesin-1 motor proteins. We find that over 95% of all breaking events are associated with the strong bending following pinning events (where the leading tip of the microtubule becomes stuck). Furthermore, the breaking rate increased exponentially with increasing curvature. These observations are explained by a model accounting for the complex mechanochemistry of a microtubule. The presence of severing enzymes is not required to observe breaking at rates comparable to those measured previously in cells.
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Citoesqueleto , Microtúbulos , Tubulina (Proteína) , Cinesinas , Ensayos de Migración Celular , Proteínas de la MembranaRESUMEN
The 3D cell migration assay was developed for the evaluation of drugs that inhibit cell migration using high throughput methods. Wound-healing assays have commonly been used for cell migration assays. However, these assays have limitations in mimicking the in vivo microenvironment of the tumor and measuring cell viability for evaluation of cell migration inhibition without cell toxicity. As an attempt to manage these limitations, cells were encapsulated with Matrigel on the surface of the pillar, and an analysis of the morphology of cells attached to the pillar through Matrigel was performed for the measurement of cell migration. The micropillar/microwell chips contained 532 pillars and wells, which measure the migration and viability of cells by analyzing the roundness and size of the cells, respectively. Cells seeded in Matrigel have a spherical form. Over time, cells migrate through the Matrigel and attach to the surface of the pillar. Cells that have migrated and adhered have a diffused shape that is different from the initial spherical shape. Based on our analysis of the roundness of the cells, we were able to distinguish between the diffuse and spherical shapes. Cells in Matrigel on the pillar that were treated with migration-inhibiting drugs did not move to the surface of the pillar and remained in spherical forms. During the conduct of experiments, 70 drugs were tested in single chips and migration-inhibiting drugs without cell toxicity were identified. Conventional migration assays were performed using transwell for verification of the four main migration-inhibiting drugs found on the chip.
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Técnicas de Cultivo de Célula , Ensayos Analíticos de Alto Rendimiento , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Ensayos de Migración Celular , Movimiento Celular , Supervivencia CelularRESUMEN
Migration is an important function for natural killer cells. Cell motility has implications in development, tissue infiltration, and cytotoxicity, and measuring the properties of natural killer (NK) cell migration using in vitro assays can be highly informative. Many researchers have an interest in studying properties of NK cell migration in the context of genetic mutation, disease, or in specific tissues and microenvironments. Motility assays can also provide information on the localization of proteins during different phases of cell migration. These assays can be performed on different surfaces for migration or coupled with chemoattractants and/or target cells to test functional outcomes or characterize cell migration speeds and phenotypes. NK cells undergo migration during differentiation in tissue, and these conditions can be modeled by culturing NK cells on a confluent bed of stromal cells on glass and imaging cell migration. Alternatively, fibronectin- or ICAM-1-coated surfaces promote NK cell migration and can be used as substrates. Here, we will describe techniques for the experimental setup and analysis of NK cell motility assays by confocal microscopy or in-incubator imaging using commercially available systems. Finally, we describe open-source software for analyzing cell migration using manual tracking or automated approaches and discuss considerations for the implementation of each of these methods.
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Células Asesinas Naturales , Células del Estroma , Ensayos de Migración Celular , Movimiento Celular/fisiología , Humanos , Microscopía ConfocalRESUMEN
Chemotaxis-directional cell movement steered by chemical gradients-involved in many biological processes including embryonic morphogenesis and immune cell function. Eukaryotic cells, in response to external gradients of attractants, use conserved mechanisms to achieve chemotaxis by regulating the actin cytoskeleton at their fronts and myosin II at their rears. Dictyostelium discoideum, an amoeba that is widely used to study chemotaxis, uses chemotaxis to move up gradients of folate to identify and locate its bacterial prey. Similarly, when starved, Dictyostelium cells synthesize and secrete cyclic AMP (cAMP) while simultaneously expressing cAMP receptors. This allows them to chemotax toward their neighbors and aggregate together. The chemotactic behavior of cells can be studied using several techniques. One such, under-agarose chemotaxis, is a robust, easy, and inexpensive assay that allows direct quantification of chemotactic parameters such as speed and directionality. With the use of high-resolution imaging, for example confocal microscopy, detailed examination of the distribution of actin and membrane proteins in migrating wild type and mutant cells can be performed. In this chapter, we describe simple and optimized methods for studying folate and cAMP chemotaxis in Dictyostelium cells under agarose.
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Dictyostelium , Ensayos de Migración Celular , Quimiotaxis/fisiología , AMP Cíclico/metabolismo , Dictyostelium/fisiología , SefarosaRESUMEN
Metformin is used to treat patients with type 2 diabetes mellitus and was found to lower the incidence of cancer. Bone metastasis is a common impairment associated with advanced breast cancer. The present study investigated the effects of metformin on human bone-derived mesenchymal stromal cells (BM-MSC)-breast cancer cell line interactions. BM-MSCs grown from box chisels were tested for growth-stimulating and migration-controlling activity on four breast cancer cell lines either untreated or after pretreatment with metformin. Growth stimulation was tested in MTT tests and migration in scratch assays. Furthermore, the expression of adipokines of BM-MSCs in response to metformin was assessed using Western blot arrays. Compared to breast cancer cell lines (3.6 ± 1.4% reduction of proliferation), 500 µM metformin significantly inhibited the proliferation of BM-MSC lines (mean 12.3 ± 2.2 reduction). Pretreatment of BM-MSCs with metformin showed variable effects of the resulting conditioned media (CM) on breast cancer cell lines depending on the specific BM-MSC-cancer line combination. Metformin significantly reduced the migration of breast cancer cell lines MDA-MB-231 and MDA-MB-436 in response to CM of drug-pretreated BM-MSCs. Assessment of metformin-induced alterations in the expression of adipokines by BM-MSC CM indicated increased osteogenic signaling and possibly impairment of metastasis. In conclusion, the anticancer activities of metformin are the result of a range of direct and indirect mechanisms that lower tumor proliferation and progression. A lower metformin-induced protumor activity of BM-MSCs in the bone microenvironment seem to contribute to the positive effects of the drug in selected breast cancer patients.
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Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Metformina/farmacología , Adipoquinas/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral/efectos de los fármacos , Ensayos de Migración Celular , Medios de Cultivo Condicionados , Humanos , Células Madre Mesenquimatosas/citología , Metástasis de la Neoplasia , Transducción de SeñalRESUMEN
BACKGROUND: Androgen receptor (AR) is an essential transcriptional factor that contributes to the development and progression of prostate cancer (PCa). NCAPD3 is a component of the condensin II complex and plays a critical role in cell mitosis by regulating chromosome condensation; however, the relationship between NCAPD3 and AR remains unknown. METHODS: Transcriptome sequencing assay is carried out to analyze the expression of the NCAP family in clinic samples. Chromatin immunoprecipitation (ChIP) sequencing, ChIP assay, and dual-luciferase assay are used to identify the androgen-responsive element in NCAPD3 enhancer. Immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, and western-blot assay are employed to check the expression of genes in PCa tissues and in PCa cells. Confocal immunofluorescence microscopy analysis is used for identifying the regulation of AR on NCAPD3-mediated chromosome condensation. Colony formation, cell cycle assay, wound healing assay, and transwell experiments are used to explore the regulation of AR on the functions of NCAPD3. In vivo experiment is employed to identify in vitro experimental results. RESULTS: NCAPD3 is an androgen/AR axis-targeted gene and is involved in AR-induced PCa cell proliferation, migration, and invasion in vitro and in vivo. Androgen treatment and AR overexpression increase the expression of NCAPD3 in PCa cell lines. The canonical exist in the enhancer region of NCAPD3. Androgen/AR axis regulates NCAPD3-invovled chromosome condensation during cell mitosis. CONCLUSIONS: Our report demonstrated that NCAPD3 is an androgen-responsive gene and upregulated by androgen/AR axis and involved in AR-promoted progression of PCa, suggesting a potential role of NCAPD3 in the PCa development.
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Proteínas de Ciclo Celular , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Adenosina Trifosfatasas/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Ensayos de Migración Celular/métodos , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Descubrimiento de Drogas , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Complejos Multiproteicos/metabolismo , Regulación hacia Arriba , Secuenciación del Exoma/métodosRESUMEN
OBJECTIVE: Skin cutaneous melanoma (SKCM) is a common cutaneous malignant tumour. This study explored the expression and the downstream regulation mechanism of guanylate binding protein 2 (GBP2), an interferon (IFN)-induced protein, in SKCM. METHODS: Western blot was employed to verify the expression of SBP2 and its downstream Wnt/ß-catenin pathway-related proteins. We studied the relationship between GBP2 and the SKCM prognosis through database analysis. In vitro, gain-and-loss-of function experiments were conducted in SKCM cells. Cell viability was monitored by the cell counting kit-8 (CCK8) assay, the colony formation assay detected cell proliferation, and apoptosis was verified by flow cytometry. Transwell assay was conducted to test cell invasion and migration, while Western blot was employed to monitor the epithelial-mesenchymal transition (EMT) of SKCM cells. RESULTS: The GBP2 expression in SKCM cells and tissues was lower than normal cells and tissues. GBP2 overexpression inhibited SKCM cell proliferation, migration, invasion, and EMT and promoted cell apoptosis. In contrast, the GBP2 knockdown had the reverse effect. Mechanically, Wnt/ß-catenin was inactivated by GBP2 overexpression and was enhanced by GBP2 knockdown. Drug activation of Wnt/ß-catenin significantly attenuated the malignant phenotypic inhibition induced by GBP2 up-regulation in SKCM cells. CONCLUSION: GBP2 exerts anti-tumour effects by inhibiting the Wnt/ß-catenin pathway in SKCM and is related to a favourable prognosis.
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Proteínas de Unión al GTP/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Melanoma , Neoplasias Cutáneas , Vía de Señalización Wnt/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ensayos de Migración Celular/métodos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Transición Epitelial-Mesenquimal/efectos de los fármacos , Técnicas de Silenciamiento del Gen/métodos , Humanos , Inmunohistoquímica , Melanoma/metabolismo , Melanoma/patología , Células Madre Neoplásicas , Pronóstico , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Melanoma Cutáneo MalignoRESUMEN
Retinoblastoma (Rb) is the most common intraocular malignant tumor in infants. Here, we investigated the function and mechanism of cyclophosphamide (CTX) in the development of Rb. Real-time quantitative polymerase chain reaction (RT-qPCR) results showed that paired box protein 5 (Pax5) expression was down-regulated in Rb tissues and cell lines. Methylation-specific PCR (MSP) results showed that the methylation level of Pax5 was up-regulated in Rb. After treatment with CTX, the Pax5 expression in Rb cell lines was increased significantly. The methylation of Pax5 and the expression of DNA methyltransferases (DNMTs) were down-regulated in the CTX group. Cyclophosphamide inhibited cell proliferation, migration, and invasion, promoted cell apoptosis via the Notch1 pathway. DNA methyltransferase inhibitor SGI-1027 had synergistic effects with CTX. Paired box protein 5 siRNA was transfected into Y79 cells treated with CTX. The expression of DNMTs, Pax5, the Notch1 pathway and apoptosis marker protein was detected by Western blotting, and changes in cell behavior were detected, respectively. Results showed that knockdown of Pax5 reversed the effects of CTX. Moreover, the Notch1 activator Valproic acid (VPA) abolished the inhibitory effects of CTX on Rb development. Moreover, CTX inhibited tumor growth in nude mice. These findings demonstrated that CTX up-regulated Pax5 expression by down-regulating DNMTs expression, and then inhibited the Notch1 signaling pathway activation and Rb growth.
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Ciclofosfamida/uso terapéutico , Factor de Transcripción PAX5/metabolismo , Receptor Notch1/metabolismo , Retinoblastoma/tratamiento farmacológico , Animales , Ensayos de Migración Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Niño , Preescolar , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Lactante , Masculino , Ratones , Neoplasias Experimentales/tratamiento farmacológico , Factor de Transcripción PAX5/genética , Receptor Notch1/genética , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Conventional cell migration assays require time-lapse imaging of live cells to trace cell migration paths, consequently demanding cumbersome hardware setup and suffering from low data throughput. In this work, we developed an assay named Tracking Cells by Footprint (TCF) based on a mechano-optical biosensor that irreversibly becomes fluorescent when sensing local cell adhesive force. Cell migration paths are visualized and recorded as fluorescent footprints on glass or elastic substrates coated with such biosensor. From the footprints, cell migration ranges, speeds and persistence are analyzed and quantified without the need of time-lapse imaging. The feasibility of TCF assays was demonstrated with three types of cells with different migratory capabilities. TCF was then applied to evaluating cell motility affected by biochemical or biomechanical cues. The results show that fibroblast motility is reduced by blebbistatin and vinblastine but promoted by bFGF (basic fibroblast growth factor), and the motility correlates with the substrate rigidity. TCF is also compatible with 96-well plates which, combined with static imaging and large-area scanning, provides high data throughput with minimal additional effort.
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Técnicas Biosensibles , Ensayos de Migración Celular , Movimiento Celular , Fibroblastos , Fenómenos MecánicosRESUMEN
ABSTRACT: The leukocytes play an important role in immune function during sepsis. We performed a retrospective study to investigate if leukocytes kinetics was associated with survival in critically ill patients with septic shock in intensive care unit (ICU).Patients with septic shock from January 1, 2014 to June 30, 2018 in our ICU were included. We extracted the demographic, clinical and laboratory data, comorbidities from our clinical database. The number of white blood cell, neutrophil and lymphocyte on day 1 and day 3 after diagnosis were collected and neutrophil to lymphocyte ratios (NLR) were calculated. Our primary outcome was 28-day mortality. Univariate and multivariate logistic regression models and cox proportional risk model were used to analyze the association between the leukocytes kinetics during first 3 days after ICU admission and the day-28 mortality.A total of 1245 septic shock patients with a 28-day mortality of 35.02% were included into analysis. There were no significant difference of lymphocyte number (0.83â±â0.02 vs 0.80â±â0.04, Pâ=â.552) between survival and non-survivals on day 1. However, the lymphocyte counts was significantly lower (0.95â±â0.03 vs 0.85â±â0.04, Pâ=â.024) on the third day. Both multivariate logistic and Cox regression analysis showed that lymphocyte counts on day 3 were associated with day-28 mortality. Moreover, Kaplan-Meier survival analysis revealed that increasing in lymphocyte counts and decreasing WBC, neutrophils and NLR during the first 3 days after diagnosis were associated with longer survival.Leukocytes kinetics during the first 3 days is a valuable prognostic marker in patients with septic shock in the ICU.
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Ensayos de Migración Celular/métodos , Recuento de Leucocitos , Linfocitos/inmunología , Neutrófilos/inmunología , Choque Séptico , China , Cuidados Críticos/métodos , Enfermedad Crítica/terapia , Femenino , Humanos , Unidades de Cuidados Intensivos/estadística & datos numéricos , Estimación de Kaplan-Meier , Recuento de Leucocitos/métodos , Recuento de Leucocitos/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Choque Séptico/diagnóstico , Choque Séptico/inmunología , Choque Séptico/mortalidadRESUMEN
OBJECTIVES: The aim of the current study was to identify the long noncoding RNAs (lncRNAs) ANRIL function and molecular pathways underlying hepatocellular carcinoma progression. METHODS: ANRIL knockdown with specific siRNA, and transfected into HepG2 cells according to the protocol of Lipofectamine 2000. Cell proliferation, apoptosis, migration and metastasis were assessed with MTT assay, flow cytometry and wound healing assay, respectively. Moreover, the expression level of ANRIL, apoptosis-related genes, and the Wnt pathway-associated genes were assessed by real time-PCR and Western blot assay. KEY FINDINGS: Knocking down of ANRIL led to alleviated cell growth and increased cell apoptosis of HepG2 cells through markedly increased expression levels of Bax and Bad. In contrast, dramatically diminished the expressions of anti-apoptotic factors including Bid and Bcl-2 in comparison to the scrambled control group (si-NC). Furthermore, ANRIL silencing resulted in an inactivated Wnt/ß-catenin pathway by suppressing key genes associated with this pathway. CONCLUSIONS: Taken together, these findings imply new insights into the regulatory network of the Wnt pathway through lncRNA ANRIL that indicate ANRIL may be a therapeutic factor potential for hepatocellular carcinoma.