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1.
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
2.
Chem Commun (Camb) ; 55(57): 8227-8230, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31268107

RESUMO

An original family of multivalent vectors encompassing gemini and facial amphiphilicity, namely cationic Siamese twin surfactants, has been prepared from the disaccharide trehalose; molecular engineering lets us modulate the self-assembling properties and the topology of the nanocomplexes with plasmid DNA for efficient gene delivery in vitro and in vivo.


Assuntos
Nanoestruturas/química , Plasmídeos/química , Tensoativos/química , Transfecção/métodos , Trealose/química , Animais , Linhagem Celular , Cercopithecus aethiops , Humanos , Camundongos , Plasmídeos/metabolismo
3.
Int J Nanomedicine ; 14: 4353-4366, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354265

RESUMO

Purpose: Gene therapy has become a promising remedy to treat disease by modifying the person's genes. The therapeutic potential of related tools such as CRISPR-Cas9 depends on the efficiency of delivery to the targeted cells. Numerous transfection reagents have been designed and lots of efforts have been devoted to develop carriers for this purpose. Therefore, the aim of the present study was to develop novel cholesterol-rich lipid-based nanoparticles to enhance transfection efficiency and serum stability. Materials and methods: We constructed two-, three- and four-component cationic liposomes (CLs) to evaluate the combined effect of cholesterol domain and DOPE (dioleoyl phosphatidylethanolamine), a fusogenic lipid, and the PEG (polyethylene glycol) moiety location inside or outside of the cholesterol domain on transfection efficiency and other properties of the particle. Lipoplex formation and pDNA (plasmid DNA) entrapment were assessed by gel retardation assay at different N/P ratios (3, 5, 7). Physicochemical characteristics, cytotoxicity, serum stability and endosomal escape capability of the lipoplexes were studied and transfection potential was measured by firefly luciferase assay. Next, HEK293 cell line stably expressing GFP was utilized to demonstrate the editing of a reporter through Cas9 and sgRNA plasmids delivery by the selected CL formula, which showed the highest transfection efficiency. Results: Among the designed CLs, the four-component formula [DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)/DOPE/cholesterol/Chol-PEG (cholesterol-polyethylene glycol)] showed the highest rate of transfection at N/P 3. Finally, transfection of Cas9/sgRNA by this formulation at N/P 3 resulted in 39% gene-editing efficiency to knockout GFP reporter. The results also show that this CL with no cytotoxicity effect can totally protect the plasmids from enzymatic degradation in serum. Conclusion: The novel PEGylated cholesterol domain lipoplex providing serum stability, higher transfection efficiency and endosomal release can be used for in vivo Cas9/sgRNA delivery and other future gene-therapy applications.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Colesterol/química , Edição de Genes , Nanopartículas/química , Transfecção/métodos , Cátions/química , Morte Celular , Colesterol/análogos & derivados , Ensaio de Desvio de Mobilidade Eletroforética , Endossomos/metabolismo , Ácidos Graxos Monoinsaturados/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lipossomos/química , Tamanho da Partícula , Fosfatidiletanolaminas/química , Plasmídeos/metabolismo , Polietilenoglicóis/química , Compostos de Amônio Quaternário/química , RNA Guia/metabolismo , Eletricidade Estática
4.
Cancer Sci ; 110(8): 2540-2548, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31162779

RESUMO

Drug resistance makes treatment difficult in cancers. The present study identifies and analyzes drug resistance-related miRNA in colorectal cancer. We established 4 types of 5-fluorouracil (5-FU)-resistant colon cancer cell lines in vitro and in vivo. We then analyzed the miRNA expression profile by miRNA array in these 4 cell lines, and identified the drug resistance-related miRNAs. We examined the expression levels of the identified miRNA in 112 colorectal tumor samples from the patients. We identified 12 possible miRNAs involved in 5-FU resistance by miRNA arrays. We then examined the relationship between miR-31, which was the most promising among them, and drug resistance. The ectopic expression of mimic miR-31 showed significant 5-FU resistance in the parental DLD-1 cells, while anti-miR-31 caused significant growth inhibition in DLD/F cells; that is, 5-FU-resistant colon cancer cell line DLD-1 under exposure to 5-FU. When we exposed high doses of 5-FU to parent or 5-FU-resistant cells, the expression levels of miR-31 were raised higher than those of controls. Notably, the expression levels of miR-31 were positively correlated with the grade of clinical stages of colorectal tumors. The protein expression levels of factors inhibiting hypoxia-inducible factor 1 were downregulated by transfection of mimic miR-31 into DLD-1 cells. This study provides evidence supporting the association of miR-31 with 5-FU drug resistance and clinical stages of colorectal tumors.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , MicroRNAs/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Transfecção/métodos
5.
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
6.
Nat Commun ; 10(1): 2212, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101808

RESUMO

In mammalian cells, double-stranded DNA breaks (DSBs) are preferentially repaired through end-joining processes that generally lead to mixtures of insertions and deletions (indels) or other rearrangements at the cleavage site. In the presence of homologous DNA, homology-directed repair (HDR) can generate specific mutations, albeit typically with modest efficiency and a low ratio of HDR products:indels. Here, we develop hRad51 mutants fused to Cas9(D10A) nickase (RDN) that mediate HDR while minimizing indels. We use RDN to install disease-associated point mutations in HEK293T cells with comparable or better efficiency than Cas9 nuclease and a 2.7-to-53-fold higher ratio of desired HDR product:undesired byproducts. Across five different human cell types, RDN variants generally result in higher HDR:indel ratios and lower off-target activity than Cas9 nuclease, although HDR efficiencies remain strongly site- and cell type-dependent. RDN variants provide precision editing options in cell types amenable to HDR, especially when byproducts of DSBs must be minimized.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Engenharia Genética/métodos , Rad51 Recombinase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reparo de DNA por Recombinação , Proteína 9 Associada à CRISPR/genética , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Células HEK293 , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas , Células K562 , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Transfecção/métodos
7.
Nat Protoc ; 14(6): 1926-1943, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31101906

RESUMO

The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8+ T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2-4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Epitopos/imunologia , Neoplasias/imunologia , Células Cultivadas , Células Dendríticas/metabolismo , Eletroporação/métodos , Epitopos/genética , Humanos , Imunoterapia/métodos , Neoplasias/terapia , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/análise , Receptores de Antígenos de Linfócitos T/imunologia , Transfecção/métodos
8.
Methods Mol Biol ; 1966: 151-161, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041745

RESUMO

The overexpression of a specific protein is a common method for investigating the specific biological function of the substance and the mechanism of action. In vivo electrotransfer has been confirmed to be one of the most reliable, efficient and cost-effective way to overexpress a protein in a select biological tissue. Typically, this technique involves a physical injection of plasmid DNA followed by electric pulses across the injection site. Here, we introduce this method that we used to transfect green fluorescent protein (GFP)-tagged PGC-1α plasmid DNA into mouse tibialis anterior (TA) muscle, which attained high transfection efficiency with no muscle damage. To quantify the transfection efficiency, we also demonstrate the visualization of plasmid DNA transfected fibers via immunohistochemical staining on muscle cross sections.


Assuntos
Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Transfecção/métodos , Animais , Eletroporação , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde , Camundongos , Plasmídeos
9.
Methods Mol Biol ; 1966: 163-173, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041746

RESUMO

Reporter assays are useful to study nuclear receptor activation and for example to evaluate the propensity of novel drug candidates to cause induction of drug-metabolizing cytochrome P450 enzymes. Here, we describe a protocol for a reverse transfection system to study the activation of human nuclear receptors constitutive androstane receptor and pregnane X receptor. The system provides long-term stability and uniformity of DNA-carrier complexes, thus avoiding the inherent variation in conventional transfection methods. Further, the system is easily adaptable for different studies. It offers reproducible and reliable results for early drug development and mechanistic studies related to nuclear receptor activation and resulting changes in gene expression.


Assuntos
Hepatócitos/metabolismo , Receptor de Pregnano X/genética , Receptores Citoplasmáticos e Nucleares/genética , Transfecção/métodos , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/metabolismo , Genes Reporter , Humanos , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo
10.
Genes Cells ; 24(7): 473-484, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31099158

RESUMO

Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine and the treatment of various diseases. Before proceeding to clinical trials, it is important to test the efficacy and safety of iPS cell-based treatments using experimental animals. The common marmoset is a new world monkey widely used in biomedical studies. However, efficient methods that could generate iPS cells from a variety of cells have not been established. Here, we report that marmoset cells are efficiently reprogrammed into iPS cells by combining RNA transfection and chemical compounds. Using this novel combination, we generate transgene integration-free marmoset iPS cells from a variety of cells that are difficult to reprogram using conventional RNA transfection method. Furthermore, we show this is similarly effective for human and cynomolgus monkey iPS cell generation. Thus, the addition of chemical compounds during RNA transfection greatly facilitates reprogramming and efficient generation of completely integration-free safe iPS cells in primates, particularly from difficult-to-reprogram cells.


Assuntos
Reprogramação Celular , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Preparações Farmacêuticas/administração & dosagem , RNA/administração & dosagem , Transfecção/métodos , Idoso , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Platirrinos
11.
Biomed Res Int ; 2019: 2761241, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016187

RESUMO

The aim of the present study was to investigate the effects of phosphorylatable nucleus localization signal linked nucleic kinase substrate short peptide (pNNS)-conjugated chitosan (pNNS-CS) mediated miR-140 and IGF-1 in both rabbit chondrocytes and cartilage defects model. pNNS-CS was combined with pBudCE4.1-IGF-1, pBudCE4.1-miR-140, and negative control pBudCE4.1 to form pDNA/pNNS-CS complexes. Then these complexes were transfected into chondrocytes or injected intra-articularly into the knee joints. High levels of IGF-1 and miR-140 expression were detected both in vitro and in vivo. Compared with pBudCE4.1 group, in vitro, the transgenic groups significantly promoted chondrocyte proliferation, increased glycosaminoglycan (GAG) synthesis, and ACAN, COL2A1, and TIMP-1 levels, and reduced the levels of nitric oxide (NO), MMP-13, and ADAMTS-5. In vivo, the exogenous genes enhanced COL2A1, ACAN, and TIMP-1 expression in cartilage and reduced cartilage Mankin score and the contents of NO, IL-1ß, TNF-α, and GAG contents in synovial fluid of rabbits, MMP-13, ADAMTS-5, COL1A2, and COL10A1 levels in cartilage. Double gene combination showed better results than single gene. This study indicate that pNNS-CS is a better gene delivery vehicle in gene therapy for cartilage defects and that miR-140 combination IGF-1 transfection has better biologic effects on cartilage defects.


Assuntos
Doenças das Cartilagens/tratamento farmacológico , Cartilagem Articular/efeitos dos fármacos , Quitosana/farmacologia , Condrócitos/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/metabolismo , MicroRNAs/metabolismo , Peptídeos/farmacologia , Animais , Doenças das Cartilagens/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Técnicas de Transferência de Genes , Humanos , Articulação do Joelho/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Óxido Nítrico/metabolismo , Coelhos , Líquido Sinovial/efeitos dos fármacos , Líquido Sinovial/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Transfecção/métodos
12.
Molecules ; 24(8)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31013863

RESUMO

When studying polyethylenimine derivatives as nonviral vectors for gene delivery, among the important issues to be addressed are high toxicity, low transfection efficiency, and nucleic acid polyplex condensation. The molecular weight of polyethylenimine, PEGylation, biocompatibility and, also, supramolecular structure of potential carrier can all influence the nucleic acid condensation behavior, polyplex size, and transfection efficiency. The main challenge in building an efficient carrier is to find a correlation between the constituent components, as well as the synergy between them, to transport and to release, in a specific manner, different molecules of interest. In the present study, we investigated the synergy between components in dynamic combinatorial frameworks formed by connecting PEGylated squalene, poly-(ethyleneglycol)-bis(3-aminopropyl) and low molecular weight polyethylenimine components to 1,3,5-benzenetrialdehyde, via reversible imine bond, applying a dynamic combinatorial chemistry approach. We report comparative structural and morphological data, DNA binding affinity, toxicity and transfection efficiency concerning the ratio of polyethylenimine and presence or absence of poly-(ethyleneglycol)-bis(3-aminopropyl) in composition of dynamic combinatorial frameworks. In vitro biological assessments have revealed the fact that nonviral vectors containing poly-(ethyleneglycol)-bis(3-aminopropyl) and the lowest amount of polyethylenimine have significant transfection efficiency at N/P 50 ratio and display insignificant cytotoxicity on the HeLa cell line.


Assuntos
Vetores Genéticos , Polietilenoglicóis , Polietilenoimina , Transfecção/métodos , Sobrevivência Celular/efeitos dos fármacos , Vetores Genéticos/química , Vetores Genéticos/farmacologia , Células HeLa , Humanos , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Polietilenoimina/química , Polietilenoimina/farmacologia
13.
BMC Res Notes ; 12(1): 225, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987672

RESUMO

OBJECTIVE: Delivery of constructs for silencing or over-expressing genes or their modified versions is a crucial step for studying neuronal cell biology. Therefore, efficient transfection is important for the success of these experimental techniques especially in post-mitotic cells like neurons. In this study, we have assessed the transfection rate, using a previously established protocol, in both primary cortical cultures and neuroblastoma cell lines. Transfection efficiencies in these preparations have not been systematically determined before. RESULTS: Transfection efficiencies obtained herein were (10-12%) for neuroblastoma, (5-12%) for primary astrocytes and (1.3-6%) for primary neurons. We also report on cell-type specific transfection efficiency of neurons and astrocytes within primary cortical cultures when applying cell-type selective transfection protocols. Previous estimations described in primary cortical or hippocampal cultures were either based on general observations or on data derived from unspecified number of biological and/or technical replicates. Also to the best of our knowledge, transfection efficiency of pure primary neuronal cultures or astrocytes cultured in the context of pure or mixed (neurons/astrocytes) population cultures have not been previously determined. The transfection strategy used herein represents a convenient, and a straightforward tool for targeted cell transfection that can be utilized in a variety of in vitro applications.


Assuntos
Astrócitos/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Plasmídeos/metabolismo , Transfecção/métodos , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Expressão Gênica , Genes Reporter , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lipídeos/química , Lipídeos/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Especificidade de Órgãos , Plasmídeos/química , Cultura Primária de Células
14.
Methods Mol Biol ; 1979: 395-406, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028650

RESUMO

The combination of single-cell RNA-seq and CRISPR allows for efficient interrogation of possibly any number of genes, only limited by the sequencing capability. Here we describe the current protocols for CRISPR screening in single cells, from cloning and virus production to generating sequencing data.


Assuntos
Sistemas CRISPR-Cas , Análise de Célula Única/métodos , Animais , Linhagem Celular , Clonagem Molecular/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Lentivirus/genética , RNA Guia/genética , Análise de Sequência de RNA/métodos , Transdução Genética/métodos , Transfecção/métodos
15.
Biomed Pharmacother ; 114: 108814, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30953817

RESUMO

BACKGROUND: Bladder cancer is one of common malignancies worldwide. lncRNAs and miRs are reported to play crucial roles in bladder cancer. We aimed to reveal the roles and mechanisms of lncRNA PEG10 and miR-134 in bladder cancer. METHODS: Transfection was used to alter PEG10, miR-134, and LRP6 expression in human bladder cancer cell lines (T24 and HT1197). Then PEG10, miR-134 and LRP6 in cells as well as tissues were analyzed by qRT-PCR. The binding effect between miR-134 and PEG10 or LRP6 was detected by luciferase reporter assay. Viability, migration, invasion and apoptosis were evaluated by CCK-8, transwell assays and flow cytometry, respectively. LRP6 and factors related with apoptosis and signal pathways (Wnt/ß-catenin and JAK/ATAT) were detected by Western blot. Finally, we established xenograft model and tumor volume and weight were both measured and weighted. RESULTS: We found the abnormal expression of PEG10, miR-134 and LRP6 in urinary bladder tissues and bladder cancer cell lines. PEG10 overexpression promoted viability, migration and invasion of T24 and HT1197 cells as well as facilitated tumor growth. PEG10 silence inhibited viability, migration and invasion, and induced apoptosis of T24 cells by upregulating miR-134 expression. PEG10 negatively regulated miR-134 expression. miR-134 targeted LRP6 and downregulated its expression. LRP6 promoted survival, migration and invasion of T24 cells and activated Wnt/ß-catenin and JAK/STAT signal pathways. CONCLUSION: lncRNA PEG10 functioned as an oncogene in bladder cancer by sponging miR-134 and thus preventing LRP6 from degradation by miR-134. LRP6 promoted bladder cancer progression probably through activating Wnt/ß-catenin and JAK/STAT signal pathways.


Assuntos
Movimento Celular/genética , Sobrevivência Celular/genética , MicroRNAs/genética , Invasividade Neoplásica/genética , RNA Longo não Codificante/genética , Neoplasias da Bexiga Urinária/genética , Idoso , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Transdução de Sinais/genética , Transfecção/métodos , Regulação para Cima/genética , Neoplasias da Bexiga Urinária/patologia
16.
Mol Biotechnol ; 61(6): 400-409, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30945164

RESUMO

Transgenic chickens are of great interest for the production of recombinant proteins in their eggs. However, the use of constitutive strong promoters or the tissue-specific ovalbumin promoter for the generation of the transgenic chickens have different drawbacks that have to be overcome in order to make chicken bioreactor an efficient production system. This prompted us to investigate the use of an alternative tissue-specific promoter, the vitellogenin promoter, which could overcome the difficulties currently found in the generation of chicken bioreactors. In the present work we establish and characterize a DNA construct consisting of a fragment of the 5´-flanking region of the chicken vitellogenin II gene cloned in a reporter vector. This construct is capable of showing the ability of the promoter to drive expression of a reporting gene in a tissue-specific manner and in a way that closely resembles physiologic regulation of vitellogenin, making it an ideal candidate to be used in the future for generation of avian bioreactors. Besides, we validate an in vitro culture system to test the performance of the DNA construct under study that could be used as a practical tool before generating any transgenic chicken. These results are important since they provide the proof of concept for the use of the vitellogenin promoter for future genetic modification of chickens bioreactors with improved characteristics in terms of quality of the recombinant protein produced.


Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Vetores Genéticos/química , Proteínas Recombinantes de Fusão/genética , Vitelogeninas/genética , Região 5'-Flanqueadora , Animais , Animais Geneticamente Modificados , Proteínas Aviárias/metabolismo , Reatores Biológicos , Embrião de Galinha , Galinhas/metabolismo , Clonagem Molecular , Estradiol/farmacologia , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Luciferases/genética , Luciferases/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Receptores Estrogênicos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodos , Vitelogeninas/metabolismo , Zigoto/efeitos dos fármacos , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
17.
ACS Appl Mater Interfaces ; 11(17): 15222-15232, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30950602

RESUMO

A gene delivery system using spiropyran as a photoswitchable photosensitizer for the controlled photochemical internalization effect was developed by engineering the outer coating of a polyethylenimine/DNA complex with a small amount of spiropyran-containing cationic copolymers. The successful binding of cationic polymers by the polyethylenimine coating was detected by the distance-sensitive fluorescence resonance energy-transfer technique that evidenced the occurrence of energy transfer between fluorescein-labeled cationic copolymers and polyethylenimine-condensed rhodamine-labeled DNA. The ternary polyplexes feature reversible controllability of singlet oxygen generation based on the dual effect of spiropyrans in photochromism and aggregation-induced enhanced photosensitization, allowing significant light-induced amplification of bPEI-mediated in vitro transgene efficiency (from original 15% to final 91%) at a low DNA dose, with the integrity of supercoiled DNA structure unaffected. The use of spiropyran without the need of other photosensitizers circumvents the issue of uncontrolled long-lasting photocytotoxicity in gene delivery.


Assuntos
Benzopiranos/química , Indóis/química , Luz , Nitrocompostos/química , Fármacos Fotossensibilizantes/química , Polietilenoimina/química , Transfecção/métodos , Sobrevivência Celular/efeitos dos fármacos , DNA/química , DNA/metabolismo , Fluoresceína/química , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Microscopia de Fluorescência , Nanopartículas/química , Nanopartículas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Rodaminas/química
18.
Prep Biochem Biotechnol ; 49(5): 453-458, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30896287

RESUMO

Existing evidence has demonstrated liposomes as the gene transporter induce the cytotoxicity during the transfection process through several known pathways. In the present study, we investigated the possibility of siRNAs targeting 3-ß-hydroxysterol △-24-reductase (DHCR24), which encodes an enzyme catalyzing the last step of cholesterol biosynthesis, to suppress the liposome cytotoxicity induced by lipid-based transfection reagent in the neuroblastoma cell line N2A. We found that the siRNAs targeting DHCR24 mRNA protect cells from the liposome-induced cell death, probably through the effect of siDHCR24s on the reduction of the cellular cholesterol and decrease in the generation of reactive oxygen species (ROS). This suggests that siRNAs targeting DHCR24 or other methods that reduce the intracellular cholesterol levels might be a good strategy for avoiding the cytotoxicity of liposomes, without impairing its efficiency of gene-delivering.


Assuntos
Sobrevivência Celular/genética , Colesterol/deficiência , Lipossomos/efeitos adversos , Proteínas do Tecido Nervoso/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Interferência de RNA , Animais , Caveolina 1/genética , Linhagem Celular Tumoral , Regulação para Baixo , Camundongos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Transfecção/métodos
19.
Methods Mol Biol ; 1953: 121-136, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912019

RESUMO

Single-domain antibodies represent an emerging class of antibody fragments with promising therapeutic and diagnostic potential. As a result, multiple strategies have been developed in order to improve their biophysical and/or biological properties. In particular, the fusion of single-domain antibodies to the Fc part of an IgG molecule has become a common protein engineering approach toward this aim. Here, we describe a detailed protocol for a streamlined laboratory-scale production of VH single-domain antibodies as Fc fusions in mammalian cells. Firstly, DNA sequence encoding VH domain of interest fused to an IgG Fc is synthesized as a double-stranded gene fragment. Secondly, the DNA fragment is directly assembled into a restriction enzyme-digested vector in an assembly reaction. Finally, vector carrying the VH-Fc-fusion construct is introduced into suspension-adapted mammalian cells for transient expression of the Fc chimeric fusion. One-week post-transfection, the expressed Fc-fusion protein is purified using protein A/G affinity chromatography. Using this protocol, we were able to clone, express, and purify milligrams of isolated anti-HER2 VH domain as a mouse IgG2c Fc fusion in less than 2 weeks. This protocol can be readily modified to express proteins of interest other than VH domains as Fc fusions.


Assuntos
Fragmentos Fc das Imunoglobulinas/genética , Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/genética , Animais , Biotinilação , Linhagem Celular , Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Camundongos , Plasmídeos/genética , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Transfecção/métodos
20.
Methods Mol Biol ; 1943: 1-25, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838606

RESUMO

Sequence-defined cationic lipo-oligomers containing unsaturated fatty acids are potent nucleic acid carriers that are produced by solid-phase supported synthesis. However, the trifluoroacetic acid (TFA)-mediated removal of acid-labile protecting groups and cleavage from the resin can be accompanied by side products caused by an addition of TFA to the double bonds of unsaturated fatty acids. These TFA adducts are converted into hydroxylated derivatives under aqueous conditions. Here we describe an optimized cleavage protocol (precooling cleavage solution to 4 °C, 20 min cleavage at 22 °C), which minimizes TFA adduct formation, retains the unsaturated hydrocarbon chain character, and ensures high yields of the synthesis.


Assuntos
Ácidos Graxos Insaturados/química , Nanopartículas/química , RNA Interferente Pequeno/genética , Transfecção/métodos , Sequência de Aminoácidos , Aminoácidos/química , Animais , Cátions/química , Linhagem Celular Tumoral , Terapia Genética/métodos , Humanos , Camundongos , Estrutura Molecular , Polimerização , Interferência de RNA , Temperatura Ambiente , Ácido Trifluoracético/química
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