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1.
Anticancer Res ; 39(9): 4737-4742, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519573

RESUMO

BACKGROUND/AIM: There are several unresolved issues regarding the combined treatment with an immune checkpoint inhibitor and anti-angiogenic agent for renal cell carcinoma (RCC) patients. The purpose of this study was to address the inhibitory effects of programmed death-ligand 1 (PD-L1) expression on growth and sensitivity to sunitinib in the mouse RCC RenCa model. MATERIALS AND METHODS: We established RenCa/sh-PD-L1 by transfecting RenCa cells with a plasmid carrying a short hairpin RNA targeted against PD-L1. The growth pattern of RenCa/sh-PD-L1 with or without sunitinib was compared to that of RenCa cells transfected with control plasmid alone (RenCa/Co). RESULTS: No significant difference in growth or sensitivity to sinitinib was noted between RenCa/sh-PD-L1 and RenCa/Co cells in vitro. The tumor volume in mice subcutaneously injected with RenCa/sh-PD-L1 was significantly smaller than that with RenCa/Co. Treatment of mice bearing each tumor with sunitinib resulted in a significant reduction of the RenCa/sh-PD-L1 tumor compared to the RenCa/Co tumor. Moreover, infiltration by CD8+ T cells of RenCa/sh-PD-L1 tumors was significantly higher than that of RenCa/Co tumors, irrespective of treatment with sunitinib. CONCLUSION: Suppressed expression of PD-L1 could increase tumor-infiltrating CD8+ T cells and result in growth inhibition as well as enhanced sensitivity to sunitinib in the RenCa model.


Assuntos
Antineoplásicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Carcinoma de Células Renais/genética , Neoplasias Renais/genética , RNA Interferente Pequeno/genética , Sunitinibe/farmacologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Expressão Gênica , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Carga Tumoral/efeitos dos fármacos
2.
Anticancer Res ; 39(9): 4811-4816, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519583

RESUMO

BACKGROUND/AIM: γ-Glutamylcyclotransferase (GGCT) is highly expressed in many forms of cancer, and is a promising therapeutic target. The present study investigated whether inhibition of GGCT enhanced the antiproliferative effects of the drug docetaxel in prostate cancer cells. MATERIALS AND METHODS: Immunohistochemistry and western blot analysis were conducted to measure GGCT expression in prostate cancer tissue samples and cell lines. GGCT was inhibited using RNAi and a novel enzymatic inhibitor, pro-GA, and cell proliferation was evaluated with single and combination treatments of GGCT inhibitors and docetaxel. RESULTS: GGCT was highly expressed in cultured prostate cancer cells and patient samples. GGCT inhibition alone inhibited prostate cancer cell line proliferation and induced cellular senescence. GGCT inhibition in combination with apoptosis-inducing docetaxel had more potent antiproliferative effects than either drug used alone. CONCLUSION: GGCT inhibition may potentiate anticancer drug efficacy.


Assuntos
Antineoplásicos/farmacologia , Docetaxel/farmacologia , Inibidores Enzimáticos/farmacologia , gama-Glutamilciclotransferase/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , gama-Glutamilciclotransferase/genética , gama-Glutamilciclotransferase/metabolismo
3.
Anticancer Res ; 39(9): 4817-4828, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519584

RESUMO

BACKGROUND/AIM: Although epidermal growth factor receptor (EGFR) is frequently activated in lung and pancreatic cancers, the efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs) is limited. Recently, brexpiprazole, an antipsychotic drug, was reported to chemosensitize glioma cells to osimertinib, a third-generation EGFR-TKI, by suppressing survivin, an anti-apoptotic protein, but their combinational effects on lung and pancreatic cancers remain unknown. The aim of this study was to examine the combinational effects of brexpiprazole and osimertinib on lung and pancreatic cancer cells in vitro and in vivo. MATERIALS AND METHODS: YM155, a suppressor of survivin, siRNA, and immunoblot were used to examine the role of survivin in osimertinib-resistance. The effect of drugs on cell viability in vitro was examined by trypan blue staining. The in vivo effects of drugs on tumor growth were examined using a xenograft mouse model. RESULTS: Brexpiprazole exerted combinational effects with osimertinib in vitro. Pharmacological and genetic suppression of survivin chemosensitized the cells to osimertinib. Moreover, the combination of brexpiprazole and osimertinib effectively suppressed tumor growth in a mouse xenograft model. CONCLUSION: Brexpiprazole is a promising drug for lung and pancreatic cancer in combination with osimertinib.


Assuntos
Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteínas Quinases/farmacologia , Quinolonas/farmacologia , Survivina/genética , Tiofenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Humanos , Masculino , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Anticancer Res ; 39(9): 4853-4864, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519588

RESUMO

BACKGROUND/AIM: Colorectal cancer (CRC) is the leading cause of cancer mortality worldwide. Its poor prognosis can be ascribed primarily to high recurrence rates. Accordingly, the aim of this study was to identify novel prognostic biomarkers and therapeutic targets for management of CRC. MATERIALS AND METHODS: To develop prognostic biomarkers, we performed RNA-seq analysis and real-time RT-PCR in primary cancer tissues with or without systemic recurrence. To characterize the molecular functions of the encoded proteins, CRC cells underexpressing or overexpressing the candidate genes were established and appropriate cell-based assays were applied. RESULTS: ITGB1 and RHOC mRNA levels were up-regulated in the recurrence group of CRC patients. Overexpression of ITGB1 or RHOC stimulated CRC cell proliferation, invasion and migration, whereas the opposite effects were observed in cells underexpressing either protein. Five-year recurrence-free survival rates were significantly higher in the ITGB1- and RHOC-underexpression groups than those in the overexpression. CONCLUSION: ITGB1 and RHOC are potential predictors of recurrence and therapeutic targets for CRC, possibly predicting a high-risk group of stage II patients.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Integrina beta1/metabolismo , Proteína de Ligação a GTP rhoC/metabolismo , Idoso , Biomarcadores Tumorais , Proliferação de Células , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Interferente Pequeno/genética , Recidiva , Análise de Sobrevida
5.
J Cancer Res Clin Oncol ; 145(10): 2433-2444, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485767

RESUMO

PURPOSE: The clinical importance of cancer stem cells (CSCs) in head and neck squamous cell carcinoma (HNSCC) is well recognized. However, a reliable method for the detection of functioning CSC has not yet been established. We hypothesized that YAP1, a transcriptional coactivator, and SOX2, a master transcription factor of SCC, may cooperatively induce stemness through transcriptional reprogramming. METHODS: We immunohistochemically examined the expression of SOX2 and YAP1 in the CD44 variant 9 (CD44v9)-positive invasion front. A CSC-inducible module was identified through a combination of siRNAs and sphere formation assays. YAP1 and SOX2 interactions were analyzed in vitro. RESULTS: The triple overexpression of SOX2, YAP1, and CD44v9 was significantly associated with poor prognosis. TCGA data revealed that the CSC-inducible module, which was related to EMT and angiogenesis, was significantly correlated with poor prognosis. The KLF7 expression, representatively chosen from the module, also correlated with poor prognosis and was essential for sphere formation and CSC propagation. Sphere stress-activated YAP1 enhanced SOX2 activity. CONCLUSIONS: The stress-triggered activation of YAP1/SOX2 transcriptionally reprograms HNSCC for the acquisition of stemness. Triple SOX2, YAP1, and CD44v9 immunostaining assays may be useful for the selection of high-risk patients with functioning CSCs, and YAP1 targeting may lead to the development of a CSC-targeting therapy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Estresse Fisiológico , Ativação Transcricional , Biomarcadores , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Neoplásicas/patologia , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Estresse Fisiológico/genética
6.
Eur J Histochem ; 63(3)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31455073

RESUMO

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.


Assuntos
Técnicas de Transferência de Genes , Lipídeos/química , Polímeros/química , RNA Interferente Pequeno/genética , Transfecção/métodos , Sequência de Bases , Carbocianinas/química , Carbocianinas/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Inativação Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Metabolismo dos Lipídeos , Lipídeos/toxicidade , Lipossomos/química , Lipossomos/metabolismo , Lipossomos/toxicidade , Neoplasias Hepáticas/genética , Polímeros/metabolismo , Polímeros/toxicidade , Interferência de RNA
7.
Anticancer Res ; 39(8): 4073-4077, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366490

RESUMO

BACKGROUND/AIM: ANRIL is a long noncoding RNA located on INK4 locus, which encodes p15 and p16 that cause G1 phase arrest in the cell cycle. ANRIL positively regulates proliferation of several kinds of cancer cells such as lung and gastric cancers. This study, examined the effect of ANRIL in head and neck squamous cell carcinoma cells. MATERIALS AND METHODS: Cells were transfected with siRNA oligonucleotides targeting ANRIL. Transfected cells were subjected to cell-cycle and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. RESULTS: Depletion of ANRIL increased p15 mRNA in FaDu cells, and p15 and p16 mRNA in CAL27 cells and inhibited proliferation of these cells. Cell cycle analysis showed that depletion of ANRIL caused arrest at the G1 phase of the cell cycle. CONCLUSION: ANRIL promotes G1 phase progression by repressing p15 and p16, and thus promotes FaDu and CAL27 cell proliferation.


Assuntos
Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , RNA Longo não Codificante/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
8.
Arch Virol ; 164(11): 2659-2669, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31385116

RESUMO

Interferon gamma (IFN-γ) is best known for its ability to regulate host immune responses; however, its direct antiviral activity is less well studied. Transmissible gastroenteritis virus (TGEV) is an economically important swine enteric coronavirus and causes acute diarrhea in piglets. At present, little is known about the function of IFN-γ in the control of TGEV infection. In this study, we demonstrated that IFN-γ inhibited TGEV infection directly in ST cells and intestine epithelial IPEC-J2 cells and that the anti-TGEV activity of IFN-γ was independent of IFN-α/ß. Moreover, IFN-γ suppressed TGEV infection in ST cells more efficiently than did IFN-α, and the combination of IFN-γ and IFN-α displayed a synergistic effect against TGEV. Mechanistically, using overexpression and functional knockdown experiments, we demonstrated that porcine interferon regulatory factor 1 (poIRF1) elicited by IFN-γ primarily mediated IFN-γ signaling cascades and the inhibition of TGEV infection by IFN-γ. Importantly, we found that TGEV elevated the expression of poIRF1 and IFN-γ in infected small intestines and peripheral blood mononuclear cells. Thus, IFN-γ plays a crucial role in curtailing enteric coronavirus infection and may serve as an effective prophylactic and/or therapeutic agent against TGEV infection.


Assuntos
Gastroenterite Suína Transmissível/imunologia , Fator Regulador 1 de Interferon/metabolismo , Interferon gama/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Animais , Linhagem Celular , Cercopithecus aethiops , Fator Regulador 1 de Interferon/genética , Interferon-alfa/imunologia , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/imunologia , Suínos , Células Vero
9.
Bioengineered ; 10(1): 306-315, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299871

RESUMO

Long non-coding RNA H19 (H19) is highly expressed in cancers and is considered to highly correlate with the extent of malignant degree. The present study was performed to determine the expression levels of H19 in anaplastic thyroid carcinoma (ATC) tissues and the role of H19 in ATC 8505C cells in vitro and in vivo. Expression of H19 was detected in 19 ATC and 19 normal thyroid tissues by real-time quantitative polymerase chain reaction. Utilizing the siRNA or short hairpin RNA (shRNA) directed against human H19 (H19 siRNA or shRNA H19) depleted H19 in ATC 8505C cells and characterized the outcomes. The results showed that H19 was overexpressed in ATC tissues. Targeting H19 inhibited proliferation, migration, and invasion and induced apoptosis in 8505C cells in vitro and inhibited tumorigenesis and metastasis in vivo. Therefore, the H19 might be an effective target for ATC molecular therapy.


Assuntos
Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Terapia de Alvo Molecular/métodos , RNA Longo não Codificante/genética , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Animais , Apoptose/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Metástase Linfática , Camundongos , Camundongos Nus , Invasividade Neoplásica , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Carcinoma Anaplásico da Tireoide/cirurgia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/cirurgia , Nódulo da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/metabolismo , Nódulo da Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/cirurgia , Tireoidectomia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Agric Food Chem ; 67(32): 8875-8883, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31347830

RESUMO

Glucan synthase (GLS) gene is known to be involved in the fungal biosynthesis of cell wall, differentiation, and growth. In the present study, a glucan synthase gene (GFGLS) in the edible mushroom Grifola frondosa with a full sequence of 5927 bp encoding a total of 1781 amino acids was cloned and characterized for the first time. GFGLSp is a membrane protein containing two large transmembrane domains connected with a hydrophilic cytoplasmic domain. With a constructed dual promoter RNA silencing vector pAN7-gfgls-dual, a GFGLS-silencing transformant iGFGLS-3 had the lowest GFGLS transcriptional expression level (26.1%) with a shorter length and thinner appearance of the mycelia, as well as decreased mycelial biomass and exo-polysaccharide production of 5.02 and 0.38 g/L, respectively. Further analysis indicated that GFGLS silence influenced slightly the monosaccharide compositions and ratios of mycelial and exo-polysaccharide. These findings suggest that GFGLS could affect mycelial growth and polysaccharide production by downregulating the glucan synthesis.


Assuntos
Polissacarídeos Fúngicos/biossíntese , Proteínas Fúngicas/metabolismo , Glucosiltransferases/metabolismo , Grifola/enzimologia , Micélio/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Glucosiltransferases/genética , Grifola/genética , Grifola/crescimento & desenvolvimento , Grifola/metabolismo , Micélio/enzimologia , Micélio/genética , Micélio/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
11.
Chem Commun (Camb) ; 55(62): 9112-9115, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31298670

RESUMO

We designed novel 4'-C-guanidinocarbohydrazidomethyl-5-methyl uridine (GMU) modified small interfering RNA (siRNA) and evaluated its biophysical and biochemical properties. Incorporation of GMU units significantly increased the thermodynamic stability as well as the enzymatic stability against nucleases in human serum. A gene silencing experiment indicated that GMU modfied siRNA (siRNA6) resulted in ≈4.9-fold more efficient knockdown than unmodified siRNA.


Assuntos
Guanidina/química , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Guanidina/análogos & derivados , Modelos Moleculares , RNA Interferente Pequeno/genética , Ribonucleases/sangue , Ribonucleases/metabolismo , Termodinâmica
12.
Gene ; 714: 143972, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31301483

RESUMO

Among cancers, lung cancer is the most morbidity and mortality disease that is remaining the fatalist. Generally, there are multiple treatment procedures for lung cancer, such as surgery, immunotherapy, radiotherapy and chemotherapy. There is, therefore, an urgent need for more specified and efficient methods for treatment of lung cancer such as RNAi, which in combination with traditional therapies could silence genes that are involved in the drug resistance. These genes may either be motivators of apoptosis inhibition, EMT and DNA repair system promoters or a member of intracellular signaling pathways, such as JAK/STAT, RAS/RAF/MEK, PI3K/AKT, NICD, B-catenin/TCF/LEF and their stimulator receptors including IGFR, EGFR, FGFR, VEGFR, CXCR4, MET, INTEGRINS, NOTCH1 and FRIZZLED, so could be considered as appropriate targets. In current review, the results of multiple studies which have employed drug application after one specific gene silencing or more than one gene from distinct pathways also simultaneous drug and RNAi usage in vitro and in vivo in lung cancer were summarized.


Assuntos
Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , RNA Interferente Pequeno/genética , Animais , Apoptose/genética , Humanos , Transdução de Sinais/genética
13.
Gene ; 714: 143997, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348981

RESUMO

Based on Akt1 and Jak1 key roles in apoptosis and proliferation of many cancers, the aim of this study was to find a new gene therapy strategy by silencing of these main anti-apoptotic genes for HNSCC treatment. Cancerous HN5 and normal HUVEC cell lines were treated with Akt1 and Jak1 siRNAs alone or with each other combined with/without cisplatin. The MTS, flow cytometry, 4',6-diamidino-2-phenylindole staining, real-time PCR and ELISA methods were utilized in this study. The highest percentage of apoptosis was observed in the treatment of Jak1 siRNA/cisplatin group in cancerous HN5 cells (96.5%) where this treatment showed 12.84% apoptosis in normal HUVEC cell line. Cell viability reduced significantly to 64.57% after treatment with Akt1 siRNA in HN5 treated group. Knocking down Akt1 and Jak1 genes using siRNAs could increase levels of apoptosis and reduce proliferation rate in HNSCC indicating the powerful effects of these genes siRNAs with or without chemotherapeutic agents in HNSCC treatment. In conclusion, the combination of siRNA-mediated gene-silencing strategy can be considered as a valuable and safe approach for sensitizing cancer cells to chemotherapeutic agents thus proposed further studies regarding this issue to approve some siRNA based therapeutics for using in clinic.


Assuntos
Apoptose/genética , Proliferação de Células/genética , Neoplasias de Cabeça e Pescoço/genética , Janus Quinase 1/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/farmacologia , Técnicas de Silenciamento de Genes/métodos , Inativação Gênica/efeitos dos fármacos , Inativação Gênica/fisiologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Células Endoteliais da Veia Umbilical Humana , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico
14.
Life Sci ; 233: 116631, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31278945

RESUMO

AIMS: Prior to reperfusion, Calpains remain inactive due to the acidic pH and elevated ionic strength in the ischemic myocardium; but Calpain is activated during myocardial reperfusion. The underlying mechanism of Calpain activation in the ischemia-reperfusion (I/R) is yet to be determined. Therefore, the present study aims to investigate the mechanism of Calpain in I/R-induced mice. MAIN METHODS: In order to detect the function of Calpain and the NLRP3/ASC/Caspase-1 axis in cardiomyocyte pyroptosis, endoplasmic reticulum (ER) stress and myocardial function, the cardiomyocytes were treated with hypoxia-reoxygenation (H/R), and NLRP3 were silenced, Calpain was overexpressed and Caspase-1 inhibitors were used to determine cardiomyocyte pyroptosis. The results obtained from the cell experiments were then verified with an animal experiment in I/R mice. KEY FINDINGS: There was an overexpression in Calpain, ASC, NLRP3, GRP78 and C/EBP homologous protein (CHOP) in cardiomyocytes following H/R. A significant increase was witnessed in lactic acid dehydrogenase (LDH) activity, cardiomyocyte pyroptosis rate, Calpain activity, reactive oxygen species (ROS) concentration, as well as activation of ER stress in cardiomyocytes after H/R. However, opposing results were observed in H/R cardiomyocytes that received siRNA Calpain, siRNA NLRP3 or Caspase-1 inhibitor treatment. Overall, the results obtained from the animal experiment were consistent with the results from the cell experiment. SIGNIFICANCE: The silencing of Calpain suppresses the activation of the NLRP3/ASC/Caspase-1 axis, thus inhibiting ER stress in mice and improving myocardial dysfunction induced by I/R, providing a novel therapeutic pathway for I/R.


Assuntos
Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Calpaína/antagonistas & inibidores , Caspase 1/química , Estresse do Retículo Endoplasmático , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Calpaína/genética , Calpaína/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Células Cultivadas , Inflamassomos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , RNA Interferente Pequeno/genética
15.
Life Sci ; 233: 116696, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31351969

RESUMO

AIMS: To explore the mechanism of how LSD1 regulates autophagy and the correlation between LSD1 and Ox-LDL-induced inflammation. MAIN METHODS: RAW264.7 cells were used during the whole study. Firstly, the effect of Ox-LDL-stimulation on LSD1 expression was detected. Through loss-of-function assay, the associations between LSD1 interference and SESN2 expression, autophagy, NLRP3 inflammasome and inflammatory cytokines were explored. Finally, the function of LSD1 exerted on activation of PI3K/Akt/mTOR signal pathway was detected using western blotting assay. KEY FINDINGS: The expression of LSD1 was significantly elevated in Ox-LDL-treated RAW264.7 cells. Inhibition of LSD1 promoted autophagy, inhibited inflammation and activated NLRP3 inflammasome. SESN2 was elevated by LSD1 inhibition, and thus activate the PI3K/Akt/mTOR signal pathway. What' more, Knockdown of SESN2 or deactivate the PI3K/Akt/mTOR signal pathway partly reversed the effect of LSD1 inhibition on autophagy. SIGNIFICANCE: Our present study drew the finding that the knockdown of LSD1 meliorated Ox-LDL-stimulated NLRP3 activation and inflammation through promoting autophagy via SESN2-mediated PI3K/Akt/mTOR pathway.


Assuntos
Autofagia , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/metabolismo , Inflamação/patologia , Lipoproteínas LDL/efeitos adversos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Nucleares/metabolismo , Animais , Células Cultivadas , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/genética , Inflamação/etiologia , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
16.
Life Sci ; 232: 116635, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31283925

RESUMO

AIMS: The pathological cardiac hypertrophy will develop into heart failure, which has no effective treatment currently. Previous studies have proved that microRNAs (miRNAs) participate in the development of cardiac hypertrophy and regulate the pathological progress. In this study, we want to investigate the role of microRNA-92b-3p (miR-92b-3p) in cardiomyocyte hypertrophy and the mechanisms involved. MATERIALS AND METHODS: Neonatal mouse ventricular cells (NMVCs) were isolated from the hearts of 1-3-d-old newborn C57BL6 mice. The isolated NMVCs were induced hypertrophic phenotype by Angiotensin-II (Ang-II) and the cell size was examined by FITC-phalloidin staining assay. The expression of miR-92b-3p was determined by quantitative real-time PCR (qRT-qPCR). MRNA and protein level of ß-MHC, ACTA1 and HAND2 in NMVCs transfected with miR-92b-3p mimic and inhibition were assessed by RT-qPCR assay and western blot assay, respectively. Dual luciferase assay was used to verify the interaction between miR-92b-3p and the 3'-untranslated region (UTR) of HAND2 gene. KEY FINDINGS: MiR-92b-3p and HAND2 were significantly increased in Ang-II-induced NMVCs. Overexpression of miR-92b-3p can ameliorate Ang-II-induced cardiomyocyte hypertrophy. MiR-92b-3p negatively regulated HAND2 expression at the transcriptional level. Both miR-92b-3p mimic and HAND2 siRNA could efficiently inhibit Ang-II-induced hypertrophy in mouse cardiomyocytes. SIGNIFICANCE: MiR-92b-3p inhibits Ang-II-induced cardiomyocyte hypertrophy via targeting HAND2.


Assuntos
Angiotensina II/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/patologia , Regiões 3' não Traduzidas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Modelos Animais de Doenças , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima
17.
Life Sci ; 232: 116637, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31288014

RESUMO

Keloid is characterized by overactive fibroblasts. Forkhead box M1 (FOXM1) is transcription factor that plays important roles in the progression of fibrosis. However, the role of FOXM1 in keloid has not been elucidated. In the present study, we examined the expression levels of FOXM1 in clinical keloid tissue specimens and primary keloid fibroblasts (KFs). The results showed that FOXM1 levels were significantly increased in both keloid tissues and KFs. To further investigate the biological functions of FOXM1, FOXM1 was knocked down in KFs by transfection with small interfering RNA targeting FOXM1 (si-FOXM1). Knockdown of FOXM1 inhibited transforming growth factor-ß1 (TGF-ß1)-induced cell proliferation and migration of KFs. Besides, the increased expressions of collagen (coll I), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) in TGF-ß1-induced KFs were suppressed by si-FOXM1 transfection. Furthermore, TGF-ß1-induced increase in p-Smad2 and p-Smad3 expressions was attenuated by FOXM1 knockdown. These data indicated that knockdown of FOXM1 inhibited TGF-ß1-induced KFs activation and extracellular matrix (ECM) accumulation, which was attributed to the inhibition of TGF-ß1/Smad pathway.


Assuntos
Proteína Forkhead Box M1/deficiência , Queloide/metabolismo , Actinas/metabolismo , Adolescente , Adulto , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Técnicas de Silenciamento de Genes/métodos , Humanos , Queloide/genética , Masculino , Fosforilação , RNA Interferente Pequeno/genética , Transdução de Sinais , Proteína Smad2/antagonistas & inibidores , Proteína Smad2/metabolismo , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/metabolismo
18.
BMC Bioinformatics ; 20(1): 340, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208316

RESUMO

BACKGROUND: As a highly efficient and specific gene regulation technology, RNAi has broad application fields and good prospects. The effect of RNAi enhances as the dosage of siRNA increases, while an exorbitant siRNA dosage will inhibit the RNAi effect. So it is crucial to formulate a dose-effect model to describe the degradation effects of the target mRNA at different siRNA dosages. RESULTS: In this work, a simple RNA interference model with hill kinetic function (Giulia Cuccato et al. (2011)) is extended. Firstly, by introducing both the degradation time delay τ1 of mRNA caused by siRNA and the transportation time delay τ2 of mRNA from the nucleus to the cytoplasm during protein translation, one acquires a novel delay differential equations (DDEs) model with physiology lags. Secondly, qualitative analyses are executed to identify regions of stability of the positive equilibrium and to determine the corresponding parameter scales. Next, the approximate period of the limit cycle at Hopf bifurcation points is computed. Furthermore we analyze the parameter sensitivity of the limit cycle. Finally, we propose an optimal strategy to select siRNA dosage which arouses significant silencing efficiency. CONCLUSIONS: Our researches indicate that when the dosage of siRNA is large, oscillating periods are identical for disparate number of siRNA target sites even if it greatly impacts the critical siRNA dosage which is the switch of oscillating behavior. Furthermore, parametric sensitivity analyses of limit cycle disclose that both of degradation lag and maximum degradation rate of mRNA due to RNAi are principal elements on determining periodic oscillation. Our explorations will provide evidence for gene regulation and RNAi.


Assuntos
Células Eucarióticas/metabolismo , Periodicidade , Interferência de RNA , Animais , Simulação por Computador , Humanos , Modelos Genéticos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética
19.
Int J Nanomedicine ; 14: 3557-3569, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31190812

RESUMO

Purpose: Combining siRNA and other chemotherapeutic agents into one nanocarrier can overcome the multidrug resistance (MDR) phenomenon by synergistically MDR relative genes silencing and elevated chemotherapeutic activity. Most of these systems are typically fabricated through complicated procedures, which involves materials preparation, drug loading and modifications. Herein, the purpose of this study is to develop a new and fast co-delivery system of siRNA and doxorubicin for potentially synergistic cancer treatment. Methods: The co-delivery system is constructed conveniently by a stable complex consisting of doxorubicin bound to siRNA via intercalation firstly, followed by interacting with (3-Aminopropyl)triethoxysilane (APTES) electrostatically and Tetraethyl orthosilicate (TEOS) co-condensed, and the characterizations of the resultant nanocarrier are also investigated. Furthermore, this study evaluates the synergistic anti-cancer efficacy in MCF-7/MDR cells after treatment of siRNA and doxorubicin 'two in one' nanocarriers. Results: We establish a new and fast method to craft a co-delivery system of siRNA and doxorubicin with controllable and nearly uniform size, and the entire fabrication process only costs in about 10 minutes. The resultant co-delivery system presents high loading capacities of siRNA and doxorubicin, and the encapsulated doxorubicin plays a pH-responsive control release. Further, biological functionality tests of the synthesized co-delivery nanocarriers show high inhibition of P-gp protein encoded by MDR-1 gene in MCF-7/MDR cells (a variant of human breast cancer cell line with drug resistance) after transfection of these nanocarriers carrying MDR-1 siRNA and doxorubicin simultaneously, which sensitize the MCF-7/MDR cells to doxorubicin, overall leading to improved cell suppression. Conclusion: Collectively, this co-delivery system not only serves as potent therapeutics for synergistic cancer therapy, it also may facilitate the bench-to-bedside translation of combinatorial delivery system as a robust drug nanocarrier by allowing for fabricating a simply and fast nanocarrier for co-delivery of siRNA and doxorubicin with predictable high production rate.


Assuntos
Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , RNA Interferente Pequeno/administração & dosagem , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Inativação Gênica , Humanos , Células MCF-7 , Nanopartículas/administração & dosagem , Nanopartículas/ultraestrutura , RNA Interferente Pequeno/genética , Dióxido de Silício/química
20.
Nat Commun ; 10(1): 2702, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221991

RESUMO

Most cationic vectors are difficult to avoid the fate of small interfering RNA (siRNA) degradation following the endosome-lysosome pathway during siRNA transfection. In this study, the endoplasmic reticulum (ER) membrane isolated from cancer cells was used to fabricate an integrative hybrid nanoplexes (EhCv/siRNA NPs) for improving siRNA transfection. Compared to the undecorated Cv/siEGFR NPs, the ER membrane-decorated EhCv/siRNA NPs exhibits a significantly higher gene silencing effect of siRNA in vitro and a better antitumor activity in nude mice bearing MCF-7 human breast tumor in vivo. Further mechanistic studies demonstrate that functional proteins on the ER membrane plays important roles on improving cellular uptake and altering intracellular trafficking pathway of siRNA. It is worth to believe that the ER membrane decoration on nanoplexes can effectively transport siRNA through the endosome-Golgi-ER pathway to evade lysosomal degradation and enhance the silencing effects of siRNA.


Assuntos
Portadores de Fármacos/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Transfecção/métodos , Animais , Linhagem Celular Tumoral , Membrana Celular , Portadores de Fármacos/efeitos adversos , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Receptores ErbB/genética , Feminino , Terapia Genética/métodos , Complexo de Golgi/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Nanopartículas/química , Neoplasias/genética , Neoplasias/terapia , RNA Interferente Pequeno/efeitos adversos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
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