RESUMO
PURPOSE: To investigate the effect of microRNA-543 (miR-543) on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of triple-negative breast cancer (TNBC) cells, and the associated mechanism. METHODS: Human breast cancer cells (MDA-MB-231, HCC1937, and MCF-7, ZR-75-1) and normal human breast epithelial cell line (MCF10A) were transfected with miR-543 mimics or inhibitor using lipofectamine 2000. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were used to determine the mRNA and protein expression levels of miR-543, actin-like protein 6A (ACTL6A), vimentin, Snail, and E-cadherin in breast cancer cells/tissue. Cell counting kit-8 (CCK-8), wound-healing, and Transwell assays were used to measure the effect of miR-543 on TNBC cell proliferation, invasion, and migration. Overall survival was determined using data from Gene Expression Omnibus (GEO) and Cancer Genome Atlas (TCGA) databases. Bioinformatics analysis and luciferase reporter gene assay were used to determine the regulatory effect of miR-543 on ACTL6A. RESULTS: The level of expression of miR-543 was significantly lower in breast cancer cells/tissue than in normal human breast epithelial cell/tissue (p < 0.05). MicroRNA-543 expression level was significantly reduced in TNBC cells/tissue, relative to the other breast cancer cells/normal breast tissue (p < 0.05). MicroRNA-543 significantly suppressed tumor growth and the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of TNBC cells, in mouse xenograft model (p < 0.05). CONCLUSIONS: miR-543 influences the biological behavior of TNBC cells by directly targeting ACTL6A gene. miR-543 could serve as a novel diagnostic and therapeutic target for TNBC.
Assuntos
Actinas/fisiologia , Movimento Celular , Proliferação de Células , Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo , Transição Epitelial-Mesenquimal , MicroRNAs/fisiologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Humanos , Camundongos , Invasividade Neoplásica , Células Tumorais CultivadasRESUMO
Neuronal protein synthesis is essential for long-term memory consolidation, and its dysregulation is implicated in various neurodegenerative disorders, including Alzheimer's disease (AD). Cellular stress triggers the activation of protein kinases that converge on the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which attenuates mRNA translation. This translational inhibition is one aspect of the integrated stress response (ISR). We found that postmortem brain tissue from AD patients showed increased phosphorylation of eIF2α and reduced abundance of eIF2B, another key component of the translation initiation complex. Systemic administration of the small-molecule compound ISRIB (which blocks the ISR downstream of phosphorylated eIF2α) rescued protein synthesis in the hippocampus, measures of synaptic plasticity, and performance on memory-associated behavior tests in wild-type mice cotreated with salubrinal (which inhibits translation by inducing eIF2α phosphorylation) and in both ß-amyloid-treated and transgenic AD model mice. Thus, attenuating the ISR downstream of phosphorylated eIF2α may restore hippocampal protein synthesis and delay cognitive decline in AD patients.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Animais , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Hipocampo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios , Cultura Primária de CélulasRESUMO
AIMS: This study aimed to investigate the effect of lymphocytes in wound healing and the underlying mechanisms, in diabetic and non-diabetic mice, using Balb/c recombination activating gene (Rag)-2 and interleukin 2 receptor gamma (IL-2Rγ) double knockout (KO) (RAG2-/- IL-2Rγ-/-) mice. MAIN METHODS: Wound healing in vivo was performed in control and STZ-induced diabetic mice, in both KO and WT mice. Inflammation and ROS production were evaluated by immunofluorescence microscopy analysis, antioxidant enzymes and angiogenesis were evaluated by quantitative PCR and immunofluorescence microscopy analysis, and wound closure kinetics evolution was evaluated by measurement of acetate tracing of the wound area. KEY FINDINGS: Wound closure was significantly delayed in KO mice, where the M1/M2 macrophage ratio and basal ROS levels were significantly increased, while antioxidant defenses and angiogenesis were significantly decreased. Moreover, the expected increase in matrix metallopeptidase (MMP)-9 protein levels in diabetic conditions was not observed in KO mice, suggesting that the mechanisms leading to the increase in MMP-9 observed in diabetic wounds may in part be lymphocyte-dependent. SIGNIFICANCE: Our results indicate that lack of lymphocytes compromises wound healing independent of diabetes. The lack of these cells, even in non-diabetic mice, mimics the phenotype observed in wounds under diabetic conditions. Moreover, the combination of diabetes and the lack of lymphocytes, further impair the wound healing conditions, indicating that when the innate regulatory function is lost in these KO mice, excessive M1 polarization, poor angiogenesis and impaired wound healing are worsen.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Subunidade gama Comum de Receptores de Interleucina/fisiologia , Linfócitos/fisiologia , Neovascularização Fisiológica/fisiologia , Cicatrização/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Inflamação/metabolismo , Subunidade gama Comum de Receptores de Interleucina/genética , Ativação de Macrófagos/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Pele/irrigação sanguínea , Pele/metabolismoRESUMO
Aim: Histone acetylation and methylation control gene expression. We investigated the impact of SET knockdown on histone methylation status and the consequences for the miRNAs levels in oral squamous cell carcinoma (OSCC). Methods: OSCC cells with and without SET knockdown were analyzed by quantitative real-time PCR to determine miRNA levels, and by immunoreactions to histone modifications. Results: The knockdown of SET increased the levels of histone H4K20me2 and miR-137. Still, SET protein binds to the miR-137 promoter region. The transfection of miR-137 mimic reduced the KI67 and Rb proteins and proliferation of OSCC cells. Conclusion: Our results show for the first time a relationship between SET and histone methylation associated with the control of miRNA expression and KI67 and Rb as targets of miR-137 in OSCC.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Chaperonas de Histonas/fisiologia , Histonas/metabolismo , MicroRNAs/genética , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Chaperonas de Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Antígeno Ki-67/metabolismo , Metilação , MicroRNAs/metabolismo , Neoplasias Bucais/patologia , Proteína do Retinoblastoma/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
Mutations in Foxn1 and Prkdc genes lead to nude and severe combined immunodeficiency (scid) phenotypes, respectively. Besides being immunodeficient, previous reports have shown that nude mice have lower gonadotropins and testosterone levels, while scid mice present increased pachytene spermatocyte (PS) apoptosis. Therefore, these specific features make them important experimental models for understanding Foxn1 and Prkdc roles in reproduction. Hence, we conducted an investigation of the testicular function in nude and scid BALB/c adult male mice and significant differences were observed, especially in Leydig cell (LC) parameters. Although the differences were more pronounced in nude mice, both immunodeficient strains presented a larger number of LC, whereas its cellular volume was smaller in comparison to the wild type. Besides these alterations in LC, we also observed differences in androgen receptor and steroidogenic enzyme expression in nude and scid mice, suggesting the importance of Foxn1 and Prkdc genes in androgen synthesis. Specifically in scid mice, we found a smaller meiotic index, which represents the number of round spermatids per PS, indicating a greater cell loss during meiosis, as previously described in the literature. In addition and for the first time, Foxn1 was identified in the testis, being expressed in LC, whereas DNA-PKc (the protein produced by Prkdc) was observed in LC and Sertoli cells. Taken together, our results show that the changes in LC composition added to the higher expression of steroidogenesis-related genes in nude mice and imply that Foxn1 transcription factor may be associated to androgen production regulation, while Prkdc expression is also important for the meiotic process.
Assuntos
Proteína Quinase Ativada por DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Células Intersticiais do Testículo/fisiologia , Células de Sertoli/fisiologia , Animais , Células Intersticiais do Testículo/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Receptores Androgênicos/metabolismo , Células de Sertoli/citologiaRESUMO
Chromatin remodeler proteins exert an important function in promoting dynamic modifications in the chromatin architecture, performing a central role in regulating gene transcription. Deregulation of these molecular machines may lead to striking perturbations in normal cell function. The CHD7 gene is a member of the chromodomain helicase DNA-binding family and, when mutated, has been shown to be the cause of the CHARGE syndrome, a severe developmental human disorder. Moreover, CHD7 has been described to be essential for neural stem cells and it is also highly expressed or mutated in a number of human cancers. However, its potential role in glioblastoma has not yet been tested. Here, we show that CHD7 is up-regulated in human glioma tissues and we demonstrate that CHD7 knockout (KO) in LN-229 glioblastoma cells suppresses anchorage-independent growth and spheroid invasion in vitro. Additionally, CHD7 KO impairs tumor growth and increases overall survival in an orthotopic mouse xenograft model. Conversely, ectopic overexpression of CHD7 in LN-428 and A172 glioblastoma cell lines increases cell motility and invasiveness in vitro and promotes LN-428 tumor growth in vivo. Finally, RNA-seq analysis revealed that CHD7 modulates a specific transcriptional signature of invasion-related target genes. Further studies should explore clinical-translational implications for glioblastoma treatment.
Assuntos
Movimento Celular , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Glioblastoma/patologia , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de NeoplasiasRESUMO
Bone fracture is a common medical condition, which may occur due to traumatic injury or disease-related conditions. Evidence suggests that microRNAs (miRNAs) can regulate osteoblast differentiation and function. In this study, we explored the effects and mechanism of miR-221 on the growth and migration of osteoblasts using MC3T3-E1 cells. The expression levels of miR-221 in the different groups were measured by qRT-PCR. Then, miR-221 mimic and inhibitor were transfected into MC3T3-E1 cells, and cell viability and migration were measured using the CCK-8 assay and the Transwell migration assay. Additionally, the expression levels of differentiation-related factors (Runx2 and Ocn) and ZFPM2 were measured by qRT-PCR. Western blot was used to measure the expression of cell cycle-related proteins, epithelial-mesenchymal transition (EMT)-related proteins, ZFPM2, and Wnt/Notch, and Smad signaling pathway proteins. miR-221 was significantly up-regulated in the patients with lumbar compression fracture (LCM) and trochanteric fracture (TF). miR-221 promoted ALP, Runx2, and OPN expressions in MC3T3-E1 cells. miR-221 overexpression significantly increased cell proliferation, migration, differentiation, and matrix mineralization, whereas suppression of miR-221 reversed these effects. Additionally, the results displayed that ZFPM2 was a direct target gene of miR-221, and overexpression of ZFPM2 reversed the promoting effects of miR-221 overexpression on osteoblasts. Mechanistic study revealed that overexpression of miR-221 inactivated the Wnt/Notch and Smad signaling pathways by regulating ZFPM2 expression. We drew the conclusions that miR-221 overexpression promoted osteoblast proliferation, migration, and differentiation by regulation of ZFPM2 expression and deactivating the Wnt/Notch and Smad signaling pathways.
Assuntos
Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fraturas Ósseas/sangue , MicroRNAs/fisiologia , Osteoblastos/fisiologia , Fatores de Transcrição/fisiologia , Células 3T3 , Análise de Variância , Animais , Western Blotting , Sobrevivência Celular/fisiologia , Proteínas de Ligação a DNA/sangue , Humanos , Camundongos , MicroRNAs/sangue , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Proteínas Smad/fisiologia , Fatores de Transcrição/sangue , Regulação para Cima/fisiologiaRESUMO
Prostate cancer (PCa) is the most common cancer among men. Metabolic syndrome (MeS) is associated with increased PCa aggressiveness and recurrence. Previously, we proposed C-terminal binding protein 1 (CTBP1), a transcriptional co-repressor, as a molecular link between these two conditions. Notably, CTBP1 depletion decreased PCa growth in MeS mice. The aim of this study was to investigate the molecular mechanisms that explain the link between MeS and PCa mediated by CTBP1. We found that CTBP1 repressed chloride channel accessory 2 (CLCA2) expression in prostate xenografts developed in MeS animals. CTBP1 bound to CLCA2 promoter and repressed its transcription and promoter activity in PCa cell lines. Furthermore, we found that CTBP1 formed a repressor complex with ZEB1, EP300 and HDACs that modulates the CLCA2 promoter activity. CLCA2 promoted PCa cell adhesion inhibiting epithelial-mesenchymal transition (EMT) and activating CTNNB1 together with epithelial marker (CDH1) induction, and mesenchymal markers (SNAI2 and TWIST1) repression. Moreover, CLCA2 depletion in PCa cells injected subcutaneously in MeS mice increased the circulating tumor cells foci compared to control. A microRNA (miRNA) expression microarray from PCa xenografts developed in MeS mice, showed 21 miRNAs modulated by CTBP1 involved in angiogenesis, extracellular matrix organization, focal adhesion and adherents junctions, among others. We found that miR-196b-5p directly targets CLCA2 by cloning CLCA2 3'UTR and performing reporter assays. Altogether, we identified a new molecular mechanism to explain PCa and MeS link based on CLCA2 repression by CTBP1 and miR-196b-5p molecules that might act as key factors in the progression onset of this disease.
Assuntos
Oxirredutases do Álcool/fisiologia , Adesão Celular/fisiologia , Canais de Cloreto/genética , Proteínas de Ligação a DNA/fisiologia , Proteína p300 Associada a E1A/fisiologia , Epigênese Genética , Transição Epitelial-Mesenquimal/fisiologia , Histona Desacetilases/fisiologia , Síndrome Metabólica/complicações , MicroRNAs/fisiologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/fisiologia , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Masculino , Camundongos , Regiões Promotoras Genéticas , Neoplasias da Próstata/complicações , Transcrição GênicaRESUMO
Bone fracture is a common medical condition, which may occur due to traumatic injury or disease-related conditions. Evidence suggests that microRNAs (miRNAs) can regulate osteoblast differentiation and function. In this study, we explored the effects and mechanism of miR-221 on the growth and migration of osteoblasts using MC3T3-E1 cells. The expression levels of miR-221 in the different groups were measured by qRT-PCR. Then, miR-221 mimic and inhibitor were transfected into MC3T3-E1 cells, and cell viability and migration were measured using the CCK-8 assay and the Transwell migration assay. Additionally, the expression levels of differentiation-related factors (Runx2 and Ocn) and ZFPM2 were measured by qRT-PCR. Western blot was used to measure the expression of cell cycle-related proteins, epithelial-mesenchymal transition (EMT)-related proteins, ZFPM2, and Wnt/Notch, and Smad signaling pathway proteins. miR-221 was significantly up-regulated in the patients with lumbar compression fracture (LCM) and trochanteric fracture (TF). miR-221 promoted ALP, Runx2, and OPN expressions in MC3T3-E1 cells. miR-221 overexpression significantly increased cell proliferation, migration, differentiation, and matrix mineralization, whereas suppression of miR-221 reversed these effects. Additionally, the results displayed that ZFPM2 was a direct target gene of miR-221, and overexpression of ZFPM2 reversed the promoting effects of miR-221 overexpression on osteoblasts. Mechanistic study revealed that overexpression of miR-221 inactivated the Wnt/Notch and Smad signaling pathways by regulating ZFPM2 expression. We drew the conclusions that miR-221 overexpression promoted osteoblast proliferation, migration, and differentiation by regulation of ZFPM2 expression and deactivating the Wnt/Notch and Smad signaling pathways.
Assuntos
Humanos , Animais , Coelhos , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , MicroRNAs/fisiologia , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fraturas Ósseas/sangue , Osteoblastos/fisiologia , Valores de Referência , Fatores de Transcrição/sangue , Sobrevivência Celular/fisiologia , Western Blotting , Análise de Variância , Células 3T3 , MicroRNAs/sangue , Proteínas de Ligação a DNA/sangueRESUMO
The transcription factor superfamily, APETALA2/ethylene response factor, is involved in plant growth and development, as well as in environmental stress responses. Here, an uncharacterized gene of this family, AtERF019, was studied in Arabidopsis thaliana under abiotic stress situations. Arabidopsis plants overexpressing AtERF019 showed a delay in flowering time of 7 days and a delay in senescence of 2 weeks when comparison with wild type plants. These plants also showed increased tolerance to water deficiency that could be explained by a lower transpiration rate, owing to their smaller stomata aperture and lower cuticle and cell wall permeability. Furthermore, using a bottom-up proteomic approach, proteins produced in response to stress, namely branched-chain-amino-acid aminotransferase 3 (BCAT3) and the zinc finger transcription factor oxidative stress 2, were only identified in plants overexpressing AtERF019. Additionally, a BCAT3 mutant was more sensitive to water-deficit stress than wild type plants. Predicted gene targets of AtERF019 were oxidative stress 2 and genes related to cell wall metabolism. These data suggest that AtERF019 could play a primary role in plant growth and development that causes an increased tolerance to water deprivation, so strengthening their chances of reproductive success.