Východiska: N-myc downstream-regulovaný gen 1 (NDRG1) má významnou funkci při progresi nádorů. U karcinomu prostaty (prostate cancer – PCa) však regulační mechanizmus NDRG1 zůstává nejasný. Materiál a metody: Hladiny exprese miR-96-5p a NDRG1 byly hodnoceny v buněčných liniích PCa a v tkáních prostaty a validovány ve veřejných databázích pomocí polymerázové řetězové reakce v reálném čase, analýzy western blot a imunohistochemie. Funkce miR-96-5p a NDRG1 byla zkoumána pomocí testů hojení ran a transwell testů in vitro a testu myšího xenoimplantátu in vivo. Dráha regulovaná pomocí NDRG1 byla testována technikou sekvenování nové generace. K detekci vztahu mezi miR-96-5p, NDRG1 a NF-kB dráhou byl použit imunofluorescenční test a test s luciferázou. Výsledky: Nadměrná exprese NDRG1 potlačuje migraci, invazi a epiteliálně-mezenchymální přechod (EMT) in vitro a inhibuje metastázy in vivo. Navíc miR-96-5p přispívá k deficitu NDRG1 a podporuje migraci a invazi buněk PCa. Kromě toho ztráta NDRG1 aktivuje dráhu NF-kB, která stimuluje fosforylaci p65 a IKBa a indukuje EMT v PCa. Závěr: MiR-96-5p podporuje migraci a invazi PCa tím, že cílí na NDRG1 a reguluje dráhu NF-kB.

Background: The N-myc downstream-regulated gene 1 (NDRG1) has been discovered as a significant gene in the progression of cancers. However, the regulatory mechanism of NDRG1 remained obscure in prostate cancer (PCa). Methods: The miR-96-5p and NDRG1 expression levels were evaluated in PCa cell lines, and prostate tissues, and validated in public databases by real-time polymerase chain reaction, western blot analysis, and immunohistochemistry. The function of miR-96-5p and NDRG1 were investigated by scratch assay and transwell assays in vitro, and mouse xenograft assay in vivo. The candidate pathway regulated by NDRG1 was conducted by the next-generation gene sequencing technique. Immunofluorescence and luciferase assays were used to detect the relation between miR-96-5p, NDRG1, and NF-kB pathway. Results: Overexpressing NDRG1 suppresses the migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro, and inhibits metastasis in vivo. Moreover, miR-96-5p contributes to NDRG1 deficiency and promotes PCa cell migration and invasion. Furthermore, NDRG1 loss activates the NF-kB pathway, which stimulates p65 and IKBa phosphorylation and induces EMT in PCa. Conclusions: MiR-96-5p promotes the migration and invasion of PCa by targeting NDRG1 and regulating the NF-kB pathway.

• Keywords
• NDRG1, miR-96-5p,
• MeSH
• Epithelial-Mesenchymal Transition MeSH
• Genetic Techniques MeSH
• Immunohistochemistry methods   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms * genetics   physiopathology   MeSH
• NF-kappa B * MeSH
• Cell Movement MeSH
• Polymerase Chain Reaction methods   MeSH
• Sequence Analysis MeSH
• Signal Transduction MeSH
• Transfection methods   MeSH
• Blotting, Western methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Clinical Study MeSH

Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology- directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.

• MeSH
• Chemical Phenomena MeSH
• CRISPR-Cas Systems * MeSH
• Gene Editing * MeSH
• Gene Expression MeSH
• Fluorescent Antibody Technique MeSH
• HEK293 Cells MeSH
• Colloids MeSH
• Humans MeSH
• Magnetite Nanoparticles * chemistry   ultrastructure   MeSH
• Plasmids genetics   MeSH
• Polyethyleneimine * chemistry   MeSH
• Genes, Reporter MeSH
• Static Electricity MeSH
• Gene Transfer Techniques * MeSH
• Transfection methods   MeSH
• Particle Size MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).

• MeSH
• Alleles MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell blood   genetics   pathology   MeSH
• Humans MeSH
• Mutation MeSH
• DNA Mutational Analysis instrumentation   methods   MeSH
• Neoplastic Cells, Circulating pathology   MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Genes, Reporter genetics   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Transfection instrumentation   methods   MeSH
• High-Throughput Nucleotide Sequencing instrumentation   methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• DNA analysis   genetics   MeSH
• Humans MeSH
• Luciferases analysis   genetics   MeSH
• Luminescent Measurements * methods   MeSH
• MCF-7 Cells MeSH
• Plasmids analysis   genetics   MeSH
• BRCA1 Protein genetics   MeSH
• Genes, Reporter MeSH
• Reproducibility of Results MeSH
• Transfection * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH

Non-viral gene delivery vectors studied in the gene therapy applications are often designed with the cationic nitrogen containing groups necessary for binding and cell release of nucleic acids. Disadvantage is a relatively high toxicity which restricts the in vivo use of such nanoparticles. Here we show, that the 3rd generation carbosilane dendrimers possessing (trimethyl)phosphonium (PMe3) groups on their periphery were able to effectively deliver the functional siRNA into the cells (B14, Cricetulus griseus), release it into the cytosol and finally to achieve up to 40% gene silencing of targeted gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) with the comparable or, in some cases, even better effectivity as their ammonium counterparts. Moreover, such cationic dendrimers show relatively low in vivo toxicity as compared to their ammonium analogues when analyzed by standard fish embryo test (FET) on Danio rerio in vivo model, with LD50 = 6.26 μM after 48 h of incubation. This is more than 10-fold improvement as compared to published values for various other types of cationic dendrimers. We discuss the potential of further increase of the transfection efficiency, endosomal escape and decrease of toxicity of such non-viral vectors, based on the systematic screening of different types of substituents on central phosphonium atom.

• MeSH
• Cell Line MeSH
• Cricetulus MeSH
• Zebrafish MeSH
• Dendrimers administration & dosage   toxicity   MeSH
• Embryo, Nonmammalian MeSH
• Lethal Dose 50 MeSH
• RNA, Small Interfering administration & dosage   MeSH
• Organophosphorus Compounds administration & dosage   toxicity   MeSH
• Silanes administration & dosage   toxicity   MeSH
• Transfection methods   MeSH
• Gene Silencing MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Conversely to common coated iron oxide nanoparticles, novel naked surface active maghemite nanoparticles (SAMNs) can covalently bind DNA. Plasmid (pDNA) harboring the coding gene for GFP was directly chemisorbed onto SAMNs, leading to a novel DNA nanovector (SAMN@pDNA). The spontaneous internalization of SAMN@pDNA into cells was compared with an extensively studied fluorescent SAMN derivative (SAMN@RITC). Moreover, the transfection efficiency of SAMN@pDNA was evaluated and explained by computational model. METHODS: SAMN@pDNA was prepared and characterized by spectroscopic and computational methods, and molecular dynamic simulation. The size and hydrodynamic properties of SAMN@pDNA and SAMN@RITC were studied by electron transmission microscopy, light scattering and zeta-potential. The two nanomaterials were tested by confocal scanning microscopy on equine peripheral blood-derived mesenchymal stem cells (ePB-MSCs) and GFP expression by SAMN@pDNA was determined. RESULTS: Nanomaterials characterized by similar hydrodynamic properties were successfully internalized and stored into mesenchymal stem cells. Transfection by SAMN@pDNA occurred and GFP expression was higher than lipofectamine procedure, even in the absence of an external magnetic field. A computational model clarified that transfection efficiency can be ascribed to DNA availability inside cells. CONCLUSIONS: Direct covalent binding of DNA on naked magnetic nanoparticles led to an extremely robust gene delivery tool. Hydrodynamic and chemical-physical properties of SAMN@pDNA were responsible of the successful uptake by cells and of the efficiency of GFP gene transfection. GENERAL SIGNIFICANCE: SAMNs are characterized by colloidal stability, excellent cell uptake, persistence in the host cells, low toxicity and are proposed as novel intelligent DNA nanovectors for efficient cell transfection.

• MeSH
• Biophysical Phenomena MeSH
• DNA chemistry   genetics   MeSH
• Genetic Vectors MeSH
• Colloids chemistry   MeSH
• Humans MeSH
• Nanoparticles chemistry   ultrastructure   MeSH
• Plasmids chemistry   genetics   MeSH
• Gene Transfer Techniques * MeSH
• Transfection methods   MeSH
• Microscopy, Electron, Transmission MeSH
• Particle Size MeSH
• Ferric Compounds chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Methods of in vivo visualization and manipulation of mitochondrial genetic machinery are limited due to the need to surpass not only the cytoplasmic membrane but also two mitochondrial membranes. Here, we employ the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mammalian mitochondria, to construct an import system for in vivo targeting of mitochondrial (mt) DNA or mtRNA, in order to provide fluorescence hybridization of the desired sequences.

• MeSH
• Hep G2 Cells MeSH
• Fluorescent Dyes MeSH
• In Situ Hybridization, Fluorescence methods   MeSH
• Humans MeSH
• DNA, Mitochondrial genetics   MeSH
• Mitochondria genetics   MeSH
• Cell Line, Tumor MeSH
• RNA, Ribosomal, 5S genetics   MeSH
• RNA genetics   MeSH
• Transfection methods   MeSH
• RNA Transport genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Polyethylenimines (PEIs) are among the most efficient polycationic non-viral transfectants. PEI architecture and size not only modulate transfection efficiency, but also cytotoxicity. However, the underlying mechanisms of PEI-induced multifaceted cell damage and death are largely unknown. Here, we demonstrate that the central mechanisms of PEI architecture- and size-dependent perturbations of integrated cellular metabolomics involve destabilization of plasma membrane and mitochondrial membranes with consequences on mitochondrial oxidative phosphorylation (OXPHOS), glycolytic flux and redox homeostasis that ultimately modulate cell death. In comparison to linear PEI, the branched architectures induced greater plasma membrane destabilization and were more detrimental to glycolytic activity and OXPHOS capacity as well as being a more potent inhibitor of the cytochrome c oxidase. Accordingly, the branched architectures caused a greater lactate dehydrogenase (LDH) and ATP depletion, activated AMP kinase (AMPK) and disturbed redox homeostasis through diminished availability of nicotinamide adenine dinucleotide phosphate (NADPH), reduced antioxidant capacity of glutathione (GSH) and increased burden of reactive oxygen species (ROS). The differences in metabolic and redox imprints were further reflected in the transfection performance of the polycations, but co-treatment with the GSH precursor N-acetyl-cysteine (NAC) counteracted redox dysregulation and increased the number of viable transfected cells. Integrated biomembrane integrity and metabolomic analysis provides a rapid approach for mechanistic understanding of multifactorial polycation-mediated cytotoxicity, and could form the basis for combinatorial throughput platforms for improved design and selection of safer polymeric vectors.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Antioxidants metabolism   pharmacology   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Cell Respiration drug effects   MeSH
• Cell Line MeSH
• Energy Metabolism drug effects   MeSH
• Glutathione metabolism   MeSH
• Homeostasis MeSH
• Kinetics MeSH
• Humans MeSH
• Mitochondrial Membranes drug effects   metabolism   MeSH
• Molecular Structure MeSH
• Molecular Weight MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress drug effects   MeSH
• Polyethyleneimine chemistry   toxicity   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Oxygen Consumption drug effects   MeSH
• Transfection methods   MeSH
• Cell Survival drug effects   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

Lectin-like transcript 1 (LLT1, gene clec2d) was identified to be a ligand for the single human NKR-P1 receptor present on NK and NK-T lymphocytes. Naturally, LLT1 is expressed on the surface of NK cells, stimulating IFN-γ production, and is up-regulated upon activation of other immune cells, e.g. TLR-stimulated dendritic cells and B cells or T cell receptor-activated T cells. While in normal tissues LLT1:NKR-P1 interaction (representing an alternative "missing-self" recognition system) play an immunomodulatory role in regulation of crosstalk between NK and antigen presenting cells, LLT1 is upregulated in glioblastoma cells, one of the most lethal tumors, where it acts as a mediator of immune escape of glioma cells. Here we report transient expression and characterization of soluble His176Cys mutant of LLT1 ectodomain in an eukaryotic expression system of human suspension-adapted HEK293S GnTI(-) cell line with uniform N-glycans. The His176Cys mutation is critical for C-type lectin-like domain stability, leading to the reconstruction of third canonical disulfide bridge in LLT1, as shown by mass spectrometry. Purified soluble LLT1 is homogeneous, deglycosylatable and forms a non-covalent homodimer whose dimerization is not dependent on presence of its N-glycans. As a part of production of soluble LLT1, we have adapted HEK293S GnTI(-) cell line to growth in suspension in media facilitating transient transfection and optimized novel high cell density transfection protocol, greatly enhancing protein yields. This transfection protocol is generally applicable for protein production within this cell line, especially for protein crystallography.

• MeSH
• Killer Cells, Natural metabolism   MeSH
• Disulfides metabolism   MeSH
• DNA metabolism   MeSH
• Glycosylation MeSH
• HEK293 Cells MeSH
• Crystallization MeSH
• Lectins, C-Type chemistry   isolation & purification   metabolism   MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Protein Multimerization MeSH
• N-Acetylglucosaminyltransferases metabolism   MeSH
• Polyethyleneimine chemistry   MeSH
• Polysaccharides metabolism   MeSH
• Solubility MeSH
• Solutions MeSH
• Protein Folding MeSH
• Amino Acid Sequence MeSH
• Protein Stability MeSH
• Protein Structure, Tertiary MeSH
• Transfection methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In mammals, double-stranded RNA (dsRNA) can mediate sequence-specific RNA interference, activate sequence-independent interferon response, or undergo RNA editing by adenosine deaminases. We showed that long hairpin dsRNA expression had negligible effects on mammalian somatic cells--expressed dsRNA was slightly edited, poorly processed into siRNAs, and it did not activate the interferon response. At the same time, we noticed reduced reporter expression in transient co-transfections, which was presumably induced by expressed dsRNA. Since transient co-transfections are frequently used for studying gene function, we systematically explored the role of expressed dsRNA in this silencing phenomenon. We demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits the expression of co-transfected reporter plasmids but not the expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent, it is found in different cell types, and it is independent of transfection method and dsRNA sequence. The inhibition occurs at the level of translation and involves protein kinase R, which binds the expressed dsRNA. Thus, dsRNA expression represents a hidden danger in transient transfection experiments and must be taken into account during interpretation of experimental results.

• MeSH
• 3T3 Cells MeSH
• RNA, Double-Stranded metabolism   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Immunoprecipitation MeSH
• Humans MeSH
• RNA, Small Interfering genetics   MeSH
• Mice MeSH
• Plasmids genetics   MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Flow Cytometry MeSH
• Gene Expression Regulation genetics   MeSH
• Genes, Reporter genetics   MeSH
• Transfection methods   MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH