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1.
Life Sci ; 248: 117469, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32109485

RESUMO

AIMS: Histone deacetylases inhibitors have shown favorable antitumor activity in clinical investigations. In the present study, we assessed the effects of a novel hydroxamic acid-based HDAC inhibitor, SB939, on breast cancer metastasis and tumor growth and characterized the underlying molecular mechanisms. MAIN METHODS: MTS, Wound-healing, and Transwell chamber invasion assays were used to detect the inhibition effects of SB939 on proliferation, migration, and invasion of breast cancer cells. Western blot, cellular immunofluorescence, and EMSA were used to explore the molecular mechanism of SB939 in suppressing breast cancer metastasis. MDA-MB-231 subcutaneous tumor-bearing model of nude mice and the spontaneous metastasis model of breast cancer were both applied to verify in vivo anti-tumor growth and anti-metastatic effects. KEY FINDINGS: Our results demonstrated that SB939 at 0.5-1 µmol/L markedly impaired the chemotactic motility of breast cancer cells. SB939 reversed epithelial-mesenchymal transition (EMT) process, as evidenced by upregulation E-cadherin expression and downregulation expressions of N-cadherin and vimentin through increasing the levels of ac-histone H3 and H4 and drecreasing the expressiongs of HDAC 5 and 4. This cascade inhibition mediated by SB939 was well interpreted by inactivating phosphorylation of STAT3, blocking its DNA-binding activity, and decreasing the expressions of STAT3-dependent target genes, including MMP2 and MMP9. Furhtermore, we found that SB939 significantly inhibited breast cancer metastasis and tumor growth in vivo and showed superior anti-tumor properties compared with SAHA in two breast cancer animal models. SIGNIFICANCE: Our findings indicate that SB939 may be an effective therapeutic option for treating advanced breast cancer.


Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Vimentina/genética , Vimentina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
2.
DNA Cell Biol ; 39(3): 459-473, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31934791

RESUMO

Lung cancer with highest morbidity and mortality seriously threatens human health worldwide. Long noncoding RNAs (lncRNAs) exert important biological functions by acting as microRNA, which is implicated in tumorigenesis and cancer development. Previous work has reported that lncRNA-ATB expression was significantly upregulated in lung adenocarcinoma tissues and promoted tumor progression; however, the mechanisms of lncRNA-ATB in lung squamous carcinoma (LSC) are still fairly elusive. In our study, lncRNA-ATB expression also markedly increases in LSC tissues and cell lines in comparison to the adjacent normal tissues and normal lung epithelial cells, respectively. Functional experiments indicate that lncRNA-ATB overexpression improves the proliferative, migratory, and invasive capabilities of normal lung epithelial cells compared with control group. Furthermore, the migratory and invasive abilities are strikingly inhibited in lncRNA-ATB silenced LSC cells. Mechanistically, lncRNA-ATB directly binds to microRNA-590-5p and downregulates microRNA-590-5p level, leading to the upregulation of NF-90 expression. In addition, lncRNA-ATB overexpression promotes the epithelial-mesenchymal transition process, where lncRNA-ATB overexpression facilitates the expression of mesenchymal phenotype related molecules N-cadherin and vimentin, while restrains the expression of epithelial phenotype related proteins E-cadherin and CK-19, compared to the control. Conversely, microRNA-590-5p mimics can reverse the results caused by lncRNA-ATB overexpression. Taken together, our initial data suggest that lncRNA-ATB overexpression may promote the progression of LSC by modulating the microRNA-590-5p/NF-90 axis.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Pulmonares/metabolismo , RNA Longo não Codificante/genética , Caderinas/genética , Caderinas/metabolismo , Carcinoma de Células Escamosas/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas do Fator Nuclear 90/genética , Proteínas do Fator Nuclear 90/metabolismo , RNA Longo não Codificante/metabolismo , Vimentina/genética , Vimentina/metabolismo
3.
Cell Physiol Biochem ; 53(4): 623-637, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31550089

RESUMO

BACKGROUND/AIMS: In articular cartilage, chondrocytes are the predominant cell type. A long-term stay in space can lead to bone loss and cartilage breakdown. Due to the poor regenerative capacity of cartilage, this may impair the crewmembers' mobility and influence mission activities. Beside microgravity other factors such as cosmic radiation and vibration might be important for cartilage degeneration. Vibration at different frequencies showed various effects on cartilage in vivo, but knowledge about its impact on chondrocytes in vitro is sparse. METHODS: Human chondrocytes were exposed to a vibration device, simulating the vibration profile occurring during parabolic flights, for 24 h (VIB) and compared to static controls. Phase-contrast microscopy, immunofluorescence, F-actin and TUNEL staining as well as quantitative real-time PCR were performed to examine effects on morphology, cell viability and shape as well as gene expression. The results were compared to earlier studies using semantic analyses. RESULTS: No morphological changes or cytoskeletal alterations were observed in VIB and no apoptotic cells were found. A reorganization and increase in fibronectin were detected in VIB samples by immunofluorescence technique. PXN, VCL, ANXA1, ANXA2, BAX, and BCL2 revealed differential regulations. CONCLUSION: Long-term VIB did not damage human chondrocytes in vitro. The reduction of ANXA2, and up-regulation of ANXA1, PXN and VCL mRNAs suggest that long-term vibration might even positively influence cultured chondrocytes.


Assuntos
Condrócitos/metabolismo , Vibração , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Linhagem Celular , Condrócitos/citologia , Condrócitos/patologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Redes Reguladoras de Genes , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Vimentina/genética , Vimentina/metabolismo
4.
Cell Physiol Biochem ; 53(4): 587-605, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31535830

RESUMO

BACKGROUND/AIMS: To investigate the role of the sympathetic nervous system (SNS) and renin-angiotensin system (RAS) in renal ischemia/reperfusion-induced (I/R) cardiac inflammatoryprofile. METHODS: Left kidney ischemia was induced in male C57BL/6 mice for 60 min, followed by reperfusion for 12 days, and treatment with or without atenolol, losartan, or enalapril. The expression of vimentin in kidney and atrial natriuretic factor (ANF) in the heart has been investigated by RT-PCR. In cardiac tissue, levels of ß1-adrenoreceptors, adenylyl cyclase, cyclic AMP-dependent protein kinase (PKA), noradrenaline, adrenaline (components of SNS), type 1 angiotensin II receptors (AT1R), angiotensinogen/Ang II and renin (components of RAS) have been measured by Western blotting and HPLC analysis. A panel of cytokines - tumour necrosis factor (TNF-α), interleukin IL-6, and interferon gamma (IFN-γ) - was selected as cardiac inflammatory markers. RESULTS: Renal vimentin mRNA levels increased by >10 times in I/R mice, indicative of kidney injury. ANF, a marker of cardiac lesion, increased after renal I/R, the values being restored to the level of Sham group after atenolol or enalapril treatment. The cardiac inflammatory profile was confirmed by the marked increase in the levels of mRNAs of TNF-α, IL-6, and IFN-γ. Atenolol and losartan reversed the upregulation of TNF-α expression, whereas enalapril restored IL-6 levels to Sham levels; both atenolol and enalapril normalized IFN-γ levels. I/R mice showed upregulation of ß1-adrenoreceptors, adenylyl cyclase, PKA and noradrenaline. Renal I/R increased cardiac levels of AT1R, which decreased after losartan or enalapril treatment. Renin expression also increased, with the upregulation returning to Sham levels after treatment with SNS and RAS blockers. Angiotensinogen/Ang II levels in heart were unaffected by renal I/R, but they were significantly decreased after treatment with losartan and enalapril, whereas increase in renin levels decreased. CONCLUSION: Renal I/R-induced cardiac inflammatory events provoked by the simultaneous upregulation of SNS and RAS in the heart, possibly underpin the mechanism involved in the development of cardiorenal syndrome.


Assuntos
Rim/metabolismo , Miocárdio/metabolismo , Sistema Renina-Angiotensina , Sistema Nervoso Simpático/metabolismo , Animais , Atenolol/farmacologia , Atenolol/uso terapêutico , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Catecolaminas/metabolismo , Enalapril/farmacologia , Enalapril/uso terapêutico , Interleucina-6/metabolismo , Losartan/farmacologia , Losartan/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Sistema Nervoso Simpático/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Vimentina/genética , Vimentina/metabolismo
5.
Biomed Pharmacother ; 118: 109253, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31545288

RESUMO

OBJECTIVE: To investigate the regulation mechanism of baicalin on triple negative breast cancer (TNBC)'s biological network by a systematic biological strategy and cytology experiment. METHODS: A systematic biological methodology is utilized to predict the potential targets of baicalin, collect the genes of TNBC, and analyze the TNBC and baicalin's network. After the systematic biological analysis is performed, the cytology experiment, real-time quantitative PCR (qPCR) is used to validate the key biological processes and signaling pathways. RESULTS: After systematic biological analysis, two networks were constructed and analyzed: (1) TNBC network; (2) Baicalin-TNBC protein-protein interaction (PPI) network. Several TNBC-related, treatment-related targets, clusters, signaling pathways and biological processes were found. Cytology experiment shows that baicalin can inhibit the proliferation, migration and invasion of breast cancer MDA-MB-231 cells in vitro (P < 0.05). The results of qPCR showed that baicalin increase the expression of E-cadherin mRNA, and decrease the expression of vimentin, ß-catenin, c-Myc and MMP-7 mRNA in LPS-induced breast cancer MDA-MB-231 cells (P < 0.05). CONCLUSION: Baicalin may achieve anti-tumor effects through regulating the targets, biological processes and pathways found in this research.


Assuntos
Flavonoides/uso terapêutico , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Flavonoides/química , Flavonoides/farmacologia , Humanos , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Invasividade Neoplásica , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vimentina/genética , Vimentina/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
6.
BMC Cancer ; 19(1): 894, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492158

RESUMO

BACKGROUNDS: Heterogeneous ribonucleoproteins (hnRNPs) are involved in the metastasis-related network. Our previous study demonstrated that hnRNP K is associated with epithelial-to-mesenchymal transition (EMT) in A549 cells. However, the precise molecular mechanism of hnRNP K involved in TGF-ß1-induced EMT remains unclear. This study aimed to investigate the function and mechanism of hnRNP K interacted with microtubule-associated protein 1B light chain (MAP 1B-LC1) in TGF-ß1-induced EMT. METHODS: Immunohistochemistry was used to detect the expression of hnRNP K in non-small-cell lung cancer (NSCLC). GST-pull down and immunofluorescence were performed to demonstrate the association between MAP 1B-LC1 and hnRNP K. Immunofluorescence, transwell assay and western blot was used to study the function and mechanism of the interaction of MAP 1B-LC1 with hnRNP K during TGF-ß1-induced EMT in A549 cells. RESULTS: hnRNP K were highly expressed in NSCLC, and NSCLC with higher expression of hnRNP K were more frequently rated as high-grade tumors with poor outcome. MAP 1B-LC1 was identified and validated as one of the proteins interacting with hnRNP K. Knockdown of MAP 1B-LC1 repressed E-cadherin downregulation, vimentin upregulation and actin filament remodeling, decreased cell migration and invasion during TGF-ß1-induced EMT in A549 cells. hnRNP K increased microtubule stability via interacting with MAP 1B-LC1 and was associated with acetylated ɑ-tubulin during EMT. CONCLUSION: hnRNP K can promote the EMT process of lung cancer cells induced by TGF-ß1 through interacting with MAP 1B-LC1. The interaction of MAP 1B/LC1 with hnRNP K may improve our understanding on the mechanism of TGF-ß1-induced EMT in lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Células A549 , Acetilação , Antígenos CD/genética , Caderinas/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/fisiologia , Pessoa de Meia-Idade , Ligação Proteica , Tubulina (Proteína)/metabolismo , Vimentina/genética
7.
Oncogene ; 38(43): 6940-6957, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31409905

RESUMO

ZFP42 zinc finger protein (REX1), a pluripotency marker in mouse pluripotent stem cells, has been identified as a tumor suppressor in several human cancers. However, the function of REX1 in cervical cancer remains unknown. Both IHC and western blot assays demonstrated that the expression of REX1 protein in cervical cancer tissue was much higher than that in normal cervical tissue. A xenograft assay showed that REX1 overexpression in SiHa and HeLa cells facilitated distant metastasis but did not significantly affect tumor formation in vivo. In addition, in vitro cell migration and invasion capabilities were also promoted by REX1. Mechanistically, REX1 overexpression induced epithelial-to-mesenchymal transition (EMT) by upregulating VIMENTIN and downregulating E-CADHERIN. Furthermore, the JAK2/STAT3-signaling pathway was activated in REX1-overexpressing cells, which also exhibited increased levels of p-STAT3 and p-JAK2, as well as downregulated expression of SOCS1, which is an inhibitor of the JAK2/STAT3-signaling pathway, at both the transcriptional and translational levels. A dual-luciferase reporter assay and qChIP assays confirmed that REX1 trans-suppressed the expression of SOCS1 by binding to two specific regions of the SOCS1 promoter. Therefore, all our data suggest that REX1 overexpression could play a crucial role in the metastasis and invasion of cervical cancer by upregulating the activity of the JAK2/STAT3 pathway by trans-suppressing SOCS1 expression.


Assuntos
Transição Epitelial-Mesenquimal/genética , Fatores de Transcrição Kruppel-Like/genética , Metástase Neoplásica/genética , Transdução de Sinais/genética , Proteína 1 Supressora da Sinalização de Citocina/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Animais , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HeLa , Humanos , Janus Quinase 2/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica/patologia , Biossíntese de Proteínas/genética , Fator de Transcrição STAT3/genética , Transcrição Genética/genética , Regulação para Cima/genética , Vimentina/genética
8.
Mol Med Rep ; 20(2): 1915-1924, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31257527

RESUMO

Kidney­type glutaminase (GLS1) plays a significant role in tumor metabolism. Our recent studies demonstrated that GLS1 was aberrantly expressed in hepatocellular carcinoma (HCC) and facilitated tumor progression. However, the roles of GLS1 in intrahepatic cholangiocarcinoma (ICC) remain largely unknown. Thus, the aim of this study was to evaluate the expression and clinical significance of GLS1 in ICC. For this purpose, combined data from the Oncomine database with those of immunohistochemistry were used to determine the expression levels of GLS1 in cancerous and non­cancerous tissues. Second, a wound­healing assay and Transwell assay were used to observe the effects of the knockdown and overexpression of GLS1 on the invasion and migration of ICC cells. We examined the associations between the expression of GLS1 and epithelial­mesenchymal transition (EMT)­related markers by western blot analysis. Finally, we examined the associations between GLS1 levels and clinicopathological factors or patient prognosis. The results revealed that GLS1 was overexpressed in different digestive system tumors, including ICC, and that GLS1 expression in ICC tissue was higher than that in peritumoral tissue. The overexpression of GLS1 in RBE cells induced metastasis and invasion. Moreover, the EMT­related markers, E­cadherin and Vimentin, were regulated by GLS1 in ICC cells. By contrast, the knockdown of GLS1 expression in QBC939 cells yielded opposite results. Clinically, a high expression of GLS1 in ICC samples negatively correlated with E­cadherin expression and positively correlated with Vimentin expression. GLS1 protein expression was associated with tumor differentiation (P=0.001) and lymphatic metastasis (P=0.029). Importantly, patients with a high GLS1 expression had a poorer overall survival (OS) and a shorter time to recurrence than patients with a low GLS1 expression. Multivariate analysis indicated that GLS1 expression was an independent prognostic indicator. On the whole, the findings of this study demonstrated that GLS1 is an independent prognostic biomarker of ICC. GLS1 facilitates ICC progression and may thus prove to be a therapeutic target in ICC.


Assuntos
Biomarcadores Tumorais/genética , Colangiocarcinoma/genética , Glutaminase/genética , Recidiva Local de Neoplasia/genética , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Colangiocarcinoma/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Prognóstico , Vimentina/genética
9.
Biofactors ; 45(5): 803-817, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31317567

RESUMO

Telomerase is a specialized reverse transcriptase/terminal transferase enzyme that adds telomeric repeat sequences at the extreme end of a newly replicated chromosome. Apart from telomere lengthening, telomerase has many extracurricular activities. Telomerase is known to regulate the expression of many genes and helps in cancer progression and epithelial-to-mesenchymal transitions (EMTs). We have previously reported that human telomerase reverse transcriptase (hTERT) regulates the expression of plasminogen activator such as urokinase-type plasminogen activator (uPA) in cancer cells following a genome-wide transcriptomic study. Here, we present data substantiating these results in terms of real-time assays, western blots, and immunofluorescence. Another aim of this study is to find out the possible mechanism by which hTERT regulates the expression of plasminogen activators. We have used some molecular biology techniques such as quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence and some assays such as wound healing assay and colony formation assay to solve this question. In this study, we show a positive association between hTERT and uPA. We also demonstrate that hTERT enhances uPA expression concomitant with EMT. Knocking down of hTERT reduces uPA expression as well as reverses EMT in cancer cells. We have also found that uPA is a transforming growth factor beta (TGF-ß)-induced protein. Our observations establish that TGF-ß-induced uPA expression is hTERT dependent.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/genética , Osteoblastos/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Telomerase/genética , Fator de Crescimento Transformador beta/farmacologia , Células A549 , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Vimentina/genética , Vimentina/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
10.
Bioengineered ; 10(1): 282-291, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31311401

RESUMO

Transforming growth factor (TGF)-ß1 plays a crucial role in the epithelial-to-mesenchymal transition (EMT) in many cancer types and in thyroid cancers. Epigallocatechin-3-gallate (EGCG), the most important ingredient in the green tea, has been reported to possess antioxidant and anticancer activities. However, the cellular and molecular mechanisms explaining its action have not been completely understood. In this study, we found that EGCG significantly suppresses EMT, invasion and migration in anaplastic thyroid carcinoma (ATC) 8505C cells in vitro by regulating the TGF-ß/Smad signaling pathways. EGCG significantly inhibited TGF-ß1-induced expression of EMT markers (E-cadherin reduction and vimentin induction) in 8505C cells in vitro. Treatment with EGCG completely blocked the phosphorylation of Smad2/3, translocation of Smad4. Taken together, these results suggest that EGCG suppresses EMT and invasion and migration by blocking TGFß/Smad signaling pathways.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Catequina/análogos & derivados , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Transdução de Sinais/efeitos dos fármacos , Células Epiteliais da Tireoide/efeitos dos fármacos , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/antagonistas & inibidores , Caderinas/genética , Caderinas/metabolismo , Catequina/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Humanos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Células Epiteliais da Tireoide/metabolismo , Células Epiteliais da Tireoide/patologia , Fator de Crescimento Transformador beta1/farmacologia , Vimentina/agonistas , Vimentina/genética , Vimentina/metabolismo
11.
Genome Res ; 29(8): 1322-1328, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31239279

RESUMO

Genome editing tools have simplified the generation of knock-in gene fusions, yet the prevalent use of gene-specific homology-directed repair (HDR) templates still hinders scalability. Consequently, realization of large-scale gene tagging requires further development of approaches to generate knock-in protein fusions via generic donors that do not require locus-specific homology sequences. Here, we combine intron-based protein trapping with homology-independent repair-based integration of a generic donor and demonstrate precise, scalable, and efficient gene tagging. Because editing is performed in introns using a synthetic exon, this approach tolerates mutations in the unedited allele, indels at the integration site, and the addition of resistance genes that do not disrupt the target gene coding sequence, resulting in easy and flexible gene tagging.


Assuntos
Edição de Genes/métodos , Genoma Humano , Íntrons , Mutagênese Insercional , Proteínas Recombinantes de Fusão/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Calnexina/genética , Calnexina/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Éxons , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Vimentina/genética , Vimentina/metabolismo
12.
PLoS Pathog ; 15(6): e1007848, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181121

RESUMO

Streptococcus agalactiae (Group B Streptococcus, GBS) normally colonizes healthy adults but can cause invasive disease, such as meningitis, in the newborn. To gain access to the central nervous system, GBS must interact with and penetrate brain or meningeal blood vessels; however, the exact mechanisms are still being elucidated. Here, we investigate the contribution of BspC, an antigen I/II family adhesin, to the pathogenesis of GBS meningitis. Disruption of the bspC gene reduced GBS adherence to human cerebral microvascular endothelial cells (hCMEC), while heterologous expression of BspC in non-adherent Lactococcus lactis conferred bacterial attachment. In a murine model of hematogenous meningitis, mice infected with ΔbspC mutants exhibited lower mortality as well as decreased brain bacterial counts and inflammatory infiltrate compared to mice infected with WT GBS strains. Further, BspC was both necessary and sufficient to induce neutrophil chemokine expression. We determined that BspC interacts with the host cytoskeleton component vimentin and confirmed this interaction using a bacterial two-hybrid assay, microscale thermophoresis, immunofluorescent staining, and imaging flow cytometry. Vimentin null mice were protected from WT GBS infection and also exhibited less inflammatory cytokine production in brain tissue. These results suggest that BspC and the vimentin interaction is critical for the pathogenesis of GBS meningitis.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Encéfalo/metabolismo , Meningites Bacterianas/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus agalactiae/metabolismo , Vimentina/metabolismo , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Encéfalo/irrigação sanguínea , Encéfalo/microbiologia , Encéfalo/patologia , Endotélio Vascular , Células HeLa , Humanos , Masculino , Meningites Bacterianas/genética , Meningites Bacterianas/patologia , Camundongos , Camundongos Mutantes , Ovinos , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/patologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidade , Vimentina/genética
13.
Mol Med Rep ; 20(2): 1418-1428, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173238

RESUMO

An increasing body of evidence has demonstrated that microRNA (miR) deregulation serves pivotal roles in tumor progression and metastasis. However, the function of miR­379 in lung cancer remains understudied, particularly in non­small cell lung cancer (NSCLC). Bioinformatics and luciferase reporter analyses confirmed that conserved helix­loop­helix ubiquitous kinase (CHUK) is a target of miR­379, which may directly bind to the 3'­untranslated region of CHUK and significantly downregulate its expression in NSCLC cells. Transwell assays were used to evaluate the role of miR­379 in cell migration and invasion, and western blotting was used to address the association between miR­379 and epithelial­mesenchymal markers, including E­cadherin, cytokeratin and Vimentin. In the present study, miR­379 expression in NSCLC tissues and cell lines was downregulated, which may be associated with the poor survival of patients with NSCLC. miR­379 may act as a tumor suppressor in NSCLC, potentially by suppressing cell growth and proliferation, delaying G1­S transition, enhancing cell apoptosis and suppressing NSCLC cell migration and invasion. Furthermore, it was also observed that CHUK may function as an oncogene, and downregulation of CHUK induced by miR­379 may partially rescue the malignant characteristics of tumors, indicating that miR­379 may be suppressed in tumorigenesis. The overexpression of miR­379 may prevent the growth of NSCLC tumors via CHUK suppression and the downstream nuclear factor­κB pathway. The results of the present study demonstrated that miR­379 may act as a tumor suppressor, and may constitute a potential biomarker and a promising therapeutic agent for the treatment for NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Quinase I-kappa B/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , NF-kappa B/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Sequência de Bases , Caderinas/genética , Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Quinase I-kappa B/metabolismo , Queratinas/genética , Queratinas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MicroRNAs/agonistas , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Transdução de Sinais , Técnicas de Cultura de Tecidos , Vimentina/genética , Vimentina/metabolismo
14.
Mol Med Rep ; 20(2): 977-984, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173256

RESUMO

Stanniocalcin 2 (STC2), a secretory glycoprotein hormone, regulates many biological processes including cell proliferation, apoptosis, tumorigenesis and atherosclerosis. However, the effect of STC2 on proliferation, migration and epithelial­mesenchymal transition (EMT) progression in human colorectal cancer (CRC) cells remains poorly understood. The expression level of STC2 was determined by quantitative real­time polymerase chain reaction (qPCR) and western blot analysis. Cell Counting Kit­8 (CCK­8) was used to detect the viability of SW480 cells. The invasion and migration of cells were identified by wound healing and Transwell assays. The mRNA and protein expression levels of ß­catenin, matrix metalloproteinase (MMP)­2, MMP­9, E­cadherin and vimentin were assessed by qPCR and western blot analysis. In the present study, it was demonstrated that STC2 was highly expressed in the CRC cell lines. After silencing of STC2, the cell viability, migration and invasion were significantly reduced. Silencing of STC2 in the CRC Sw480 cells increased the expression of E­cadherin and decreased the expression of vimentin, MMP­2 and MMP­9, compared to those in the normal and empty vector group. Furthermore, the expression of ß­catenin in the STC2 gene silenced group was suppressed, and the expression of ß­catenin was reversed by Wnt activator, SB216763. These results demonstrated that STC2 participates in the development and progression of CRC by promoting CRC cell proliferation, survival and migration and activating the Wnt/ß­catenin signaling pathway.


Assuntos
Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , beta Catenina/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Células HCT116 , Células HT29 , Humanos , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Maleimidas/farmacologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Vimentina/genética , Vimentina/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
15.
J Dairy Sci ; 102(8): 7522-7535, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31155243

RESUMO

The liver becomes resistant to growth hormone before parturition in dairy cows (uncoupling of the somatotropic axis). However, the mechanism of growth hormone insensitivity has not been fully described. The aim of the present study was to improve a previous model of adult bovine hepatocytes in a sandwich culture system to ensure growth hormone receptor (GHR) expression. First, we modified the protocol for hepatocyte retrieval and tested the effect of short (18 min) and long (up to 30 min) warm ischemia on hepatocyte viability. Second, we used medium additives that affect GHR expression in vivo-insulin (INS), dexamethasone (DEX), both (INS+DEX), or no hormone additives (CTRL)-to ensure the functionality of hepatocytes, as measured by lactate dehydrogenase activity and urea concentration in the medium. We also used reverse transcriptase PCR of hepatocytes to evaluate expression of albumin (ALB), hepatocyte nuclear factor 4α (HNF4A), nuclear factor-κ-B-inhibitor α (NFKBIA), cytosolic phosphoenolpyruvate carboxykinase (PCK1), and vimentin (VIM) mRNA. Moreover, we analyzed the expression of GHRtot (GHR), GHR1A, insulin-like growth factor-1 (IGF1), and IGF binding protein-2 (IGFBP2) in response to exposure to media with the different compositions. Modification of the protocol (changes in rinsing and perfusion times, buffer composition, and the volume and standardization of collagenase) led to increased cell counts and cell viability. Short warm ischemia with the modified protocol significantly increased cell count (4.7 × 107 ± 1.9 × 107 vs. 3.5 × 106 ± 1.5 × 106 vital cells/g of liver) and viability (79.1 ± 8.4 vs. 37.1 ± 8.9%). Therefore, we gathered hepatocytes from the liver after short warm ischemia with the modified protocol. For these hepatocytes, lactate dehydrogenase activity was lower in media with INS and with DEX than in media with INS+DEX or CTRL; urea concentrations were highest at d 4 for INS+DEX. As well, HNF4A and ALB were more highly expressed in hepatocytes cultured with INS and INS+DEX than in those cultured with DEX or CTRL, and the substitution of DEX suppressed VIM and NFKBIA expression but increased PCK1 expression. The expression of GHR, GHR1A, and IGF1 was suppressed by dexamethasone (DEX and INS+DEX), whereas INS distinctly increased GHR, GHR1A, and IGF1 mRNA expression. Hepatocytes in a sandwich culture showed influenceable GHR expression; this study provides a model that can be used in studies examining factors that influence the expression and signal transduction of GHR in dairy cows.


Assuntos
Bovinos/genética , Hepatócitos/metabolismo , Fígado/citologia , Receptores da Somatotropina/genética , Animais , Bovinos/metabolismo , Células Cultivadas , Dexametasona/farmacologia , Feminino , Hormônio do Crescimento/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Gravidez , Cultura Primária de Células , Receptores da Somatotropina/metabolismo , Vimentina/genética , Vimentina/metabolismo
16.
Int J Mol Sci ; 20(11)2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31181623

RESUMO

The prognostic and therapeutic values of fibronectin have been reported in patients with renal cell carcinoma (RCC). However, the underlying mechanisms of malignancy in RCC are not completely understood. We found that silencing of fibronectin expression attenuated human RCC 786-O and Caki-1 cell growth and migration. Silencing of potential fibronectin receptor integrin α5 and integrin ß1 decreased 786-O cell ability in movement and chemotactic migration. Biochemical examination revealed a reduction of cyclin D1 and vimentin expression, transforming growth factor-ß1 (TGF-ß1) production, as well as Src and Smad phosphorylation in fibronectin-silenced 786-O and Caki-1 cells. Pharmacological inhibition of Src decreased 786-O cell growth and migration accompanied by a reduction of cyclin D1, fibronectin, vimentin, and TGF-ß1 expression, as well as Src and Smad phosphorylation. In 786-O cells, higher activities in cell growth and migration than in Caki-1 cells were noted, along with elevated fibronectin and TGF-ß1 expression. The additions of exogenous fibronectin and TGF-ß1 promoted Caki-1 cell growth and migration, and increased cyclin D1, fibronectin, vimentin, and TGF-ß1 expression, as well as Src and Smad phosphorylation. These findings highlight the role of fibronectin in RCC cell growth and migration involving Src and TGF-ß1 signaling.


Assuntos
Carcinoma de Células Renais/metabolismo , Movimento Celular , Proliferação de Células , Fibronectinas/metabolismo , Neoplasias Renais/metabolismo , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Fibronectinas/genética , Humanos , Integrina alfa5/genética , Integrina alfa5/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismo , Vimentina/genética , Vimentina/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo
17.
Reprod Biol ; 19(2): 133-138, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31080158

RESUMO

Chemotherapy may result in ovarian atrophy, a depletion of the primordial follicle pool, diminished ovarian weight, cortical and stromal fibrosis. Imatinib mesylate is an anticancer agent that inhibits competitively several receptor tyrosine kinases (RTKs). RTKs play important roles in cell metabolism, proliferation, and apoptosis. In clinic, imatinib mesylate is also known as an anti-fibrotic medicine. In the present study, the impact of imatinib on the ovarian tissue was investigated by assessing ovarian tissue fibrosis in postnatal rat administered with or without imatinib for three days. Fibrosis in the ovarian tissue was determined by histology (Picrosirius and Masson's trichrome staining) and the protein expression of vimentin and alpha-smooth muscle actin (α-SMA). Furthermore, mRNA expression of Forkhead box transcription factor O1 and O3 (FOXO1 and FOXO3), which are markers of cell proliferation was quantified. A short-term exposure to imatinib showed to increase tissue fibrosis in ovaries. This was observed by Masson's trichrome staining. Exposure to imatinib led also to a down-regulation of vimentin protein expression and up-regulation mRNA expression of FOXO3. This may indicate a role of FOXO3 in ovarian tissue fibrosis in postnatal rat ovaries.


Assuntos
Fibrose/tratamento farmacológico , Mesilato de Imatinib/farmacologia , Doenças Ovarianas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Actinas/genética , Actinas/metabolismo , Animais , Feminino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Vimentina/genética , Vimentina/metabolismo
18.
Cells ; 8(5)2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31126068

RESUMO

Intermediate filament (IF) proteins make up the largest family of cytoskeletal proteins in metazoans, and are traditionally known for their roles in fostering structural integrity in cells and tissues. Remarkably, individual IF genes are tightly regulated in a fashion that reflects the type of tissue, its developmental and differentiation stages, and biological context. In cancer, IF proteins serve as diagnostic markers, as tumor cells partially retain their original signature expression of IF proteins. However, there are also characteristic alterations in IF gene expression and protein regulation. The use of high throughput analytics suggests that tumor-associated alterations in IF gene expression have prognostic value. Parallel research is also showing that IF proteins directly and significantly impact several key cellular properties, including proliferation, death, migration, and invasiveness, with a demonstrated impact on the development, progression, and characteristics of various tumors. In this review, we draw from recent studies focused on three IF proteins most associated with cancer (keratins, vimentin, and nestin) to highlight how several "hallmarks of cancer" described by Hanahan and Weinberg are impacted by IF proteins. The evidence already in hand establishes that IF proteins function beyond their classical roles as markers and serve as effectors of tumorigenesis.


Assuntos
Carcinogênese/metabolismo , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Queratinas/metabolismo , Metástase Neoplásica/fisiopatologia , Nestina/metabolismo , Vimentina/metabolismo , Animais , Carcinogênese/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata , Inflamação/imunologia , Inflamação/metabolismo , Queratinas/genética , Queratinas/imunologia , Camundongos , Metástase Neoplásica/genética , Neovascularização Patológica/metabolismo , Nestina/genética , Vimentina/genética
19.
Front Biosci (Landmark Ed) ; 24: 1316-1329, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31136981

RESUMO

Blood-testis barrier (BTB) that is constructed by testicular Sertoli cells (SCs) is essential for spermatogenesis. Krüppel-like factor 6 (Klf6), a nuclear transcription regulator, is reported to be associated with tight junction molecules of BTB between SCs during spermatogenesis; however, the specific regulatory role and mechanism of Klf6 in BTB regulation are still unknown. Here, we primarily confirmed the temporal and spatial expression patterns of Klf6 in mouse testes. Then, Klf6 was silenced in mouse cultured SCs using either Klf6-siRNA or Klf6-shRNA lentivirus. We mainly found that: (i) Klf6 was indispensable for the proliferative activity of mouse SCs; (ii) Klf6 regulated the integrity and permeability of BTB; (iii) Klf6 knockdown led to the significant upregulation of Zo-1, Claudin-11 and Vimentin, and downregulation of Claudin-3. Furthermore, Zo-1 and Claudin-3, participated in the tight junction remolding, were determined as targets of transcription factor Klf6 by luciferase assay. In summary, our findings suggest that Klf6 regulates the BTB assembly and disassembly via mainly targeting Zo-1 and Claudin-3 in mouse SCs.


Assuntos
Barreira Hematotesticular/metabolismo , Fator 6 Semelhante a Kruppel/metabolismo , Células de Sertoli/metabolismo , Testículo/metabolismo , Junções Íntimas/metabolismo , Animais , Células Cultivadas , Claudina-3/genética , Claudina-3/metabolismo , Claudinas/genética , Claudinas/metabolismo , Regulação da Expressão Gênica , Fator 6 Semelhante a Kruppel/genética , Masculino , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Transmissão , Interferência de RNA , Espermatogênese/genética , Testículo/ultraestrutura , Vimentina/genética , Vimentina/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
20.
Int J Mol Sci ; 20(10)2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31137617

RESUMO

Liver regeneration plays a significant role in protecting liver function after liver injury or chronic liver disease. Long non-coding RNAs (lncRNAs) are considered to be involved in the proliferation of hepatocytes and liver regeneration. Therefore, this study aimed to explore the effects of LncRNA-Dreh on the regulation of hepatic progenitor cells (HPCs) during liver regeneration in rats. Initially, the rat model of liver injury was established to investigate the effect of LncRNA-Dreh down-regulation on liver tissues of rats with liver injury. Subsequently, HPCs line WB-F344 cells were transfected with interference plasmid of LncRNA-Dreh and the expression of LncRNA-Dreh and Vimentin was detected. The proliferation and migration ability of WB-F344 cells, as well as the content of albumin (ALB) and alpha fetoprotein (AFP) in cell differentiation were then determined. Disorderly arranged structure of liver tissue, a large number of HPCs set portal area as center extended to hepatic lobule and ductular reaction were observed in liver tissues of rats with liver injury. The expression of LncRNA-Dreh decreased while Vimentin increased in liver tissues of rats with liver injury. Moreover, the proliferation and migration ability, expression of Vimentin and AFP in WB-F344 cells were increased after silencing of LncRNA-Dreh and ALB was decreased. Collectively, our findings demonstrate that inhibition of LncRNA-Dreh can enhance the proliferation and migration abilities of HPCs in liver regeneration but cause abnormal differentiation of HPCs.


Assuntos
Movimento Celular , Proliferação de Células , Hepatócitos/metabolismo , Regeneração Hepática , RNA Longo não Codificante/genética , Células-Tronco/metabolismo , Animais , Linhagem Celular , Hepatócitos/fisiologia , Masculino , RNA Longo não Codificante/metabolismo , Ratos , Ratos Sprague-Dawley , Células-Tronco/fisiologia , Vimentina/genética , Vimentina/metabolismo
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