Modified siRNA effectively silence inducible immunoproteasome subunits in NSO cells

© 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.The pathogenesis of autoimmune and neurodegenerative diseases involves overexpression of inducible subunits of the immunoproteasome. However, the clinical application of inhibitors to inducible subunits of the immunoproteasome has been limited due to systemic toxicity. Here, we designed siRNAs that efficiently silence LMP2, LMP7 and MECL-1 gene expression. Inducible subunits of the immunoproteasome are complex siRNA targets because they have a long half-life; therefore, we introduced 2′-O-methyl modifications into nuclease-sensitive sites. This led to 90-95% silencing efficiency and prolonged silencing, eliminating the need for multiple transfections. Furthermore, we showed that in the absence of transfection reagent, siRNAs with lipophilic residues were able to penetrate cells more effectively and decrease the expression of inducible immunoproteasome subunits by 35% after 5 days. These results show that siRNA targeted to inducible immunoproteasome subunits have great potential for the development of novel therapeutics for autoimmune and neurodegenerative diseases

The reaction of adsorption of microorganisms by epithelial cells in smokers before and after applying an innovative dental spray with a mucoprotector

The purpose of the study. To evaluate the change in the local immunity of the oral cavity by the reaction of adsorption of microorganisms by epithelial cells in smokers before and after applying an innovative dental spray with a mucoprotector.Цель исследования – оценить изменение местного иммунитета полости рта по реакции адсорбции микроорганизмов эпителиальными клетками у курильщиков до и после нанесения инновационного стоматологического спрея с мукопротектором

Modeling of antigenomic therapy of mitochondrial diseases by mitochondrially addressed RNA targeting a pathogenic point mutation in mitochondrial DNA

Defects in mitochondrial genome can cause a wide range of clinical disorders, mainly neuromuscular diseases. Presently, no efficient therapeutic treatment has been developed against this class of pathologies. Because most of deleterious mitochondrial mutations are heteroplasmic, meaning that wild type and mutated forms of mitochondrial DNA (mtDNA) coexist in the same cell, the shift in proportion between mutant and wild type molecules could restore mitochondrial functions. Recently, we developed mitochondrial RNA vectors that can be used to address anti-replicative oligoribonucleotides into human mitochondria and thus impact heteroplasmy level in cells bearing a large deletion in mtDNA. Here, we show that this strategy can be also applied to point mutations in mtDNA. We demonstrate that specifically designed RNA molecules containing structural determinants for mitochondrial import and 20-nucleotide sequence corresponding to the mutated region of mtDNA, are able to anneal selectively to the mutated mitochondrial genomes. After being imported into mitochondria of living human cells in culture, these RNA induced a decrease of the proportion of mtDNA molecules bearing a pathogenic point mutation in the mtDNA ND5 gene

A central fragment of ribosomal protein S26 containing the eukaryote-specific motif YxxPKxYxK is a key component of the ribosomal binding site of mRNA region 5′ of the E site codon

The eukaryotic ribosomal protein S26e (rpS26e) lacking eubacterial counterparts is a key component of the ribosomal binding site of mRNA region 5′ of the codon positioned at the exit site. Here, we determined the rpS26e oligopeptide neighboring mRNA on the human 80S ribosome using mRNA analogues bearing perfluorophenyl azide-derivatized nucleotides at designed locations. The protein was cross-linked to mRNA analogues in specific ribosomal complexes, in which the derivatized nucleotide was located at positions −3 to −9. Digestion of cross-linked rpS26e with various specific proteolytic agents followed by identification of the resulting modified oligopeptides made it possible to map the cross-links to fragment 60–71. This fragment contains the motif YxxPKxYxK conserved in eukaryotic but not in archaeal rpS26e. Analysis of X-ray structure of the Tetrahymena thermophila 40S subunit showed that this motif is not implicated in the intraribosomal interactions, implying its involvement in translation process in a eukaryote-specific manner. Comparison of the results obtained with data on positioning of ribosomal ligands on the 40S subunit lead us to suggest that this motif is involved in interaction with both the 5′-untranslated region of mRNA and the initiation factor eIF3 specific for eukaryotes, providing new insights into molecular mechanisms of translation in eukaryotes

Small Interfering RNA Targeted to IGF-IR Delays Tumor Growth and Induces Proinflammatory Cytokines in a Mouse Breast Cancer Model

Insulin-like growth factor I (IGF-I) and its type I receptor (IGF-IR) play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs) for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2′-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD). Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2′-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines

Characteristic of the active substance of the Saccharomyces cerevisiae preparation having radioprotective properties

The paper describes some biological features of the radioprotective effect of double-stranded RNA preparation. It was found that yeast RNA preparation has a prolonged radioprotective effect after irradiation by a lethal dose of 9.4 Gy. 100 % of animals survive on the 70th day of observation when irradiated 1 hour or 4 days after 7 mg RNA preparation injection, 60 % animals survive when irradiated on day 8 or 12. Time parameters of repair of double-stranded breaks induced by gamma rays were estimated. It was found that the injection of the RNA preparation at the time of maximum number of double-stranded breaks, 1 hour after irradiation, reduces the efficacy of radioprotective action compared with the injection 1 hour before irradiation and 4 hours after irradiation. A comparison of the radioprotective effect of the standard radioprotector B-190 and the RNA preparation was made in one experiment. It has been established that the total RNA preparation is more efficacious than B-190. Survival on the 40th day after irradiation was 78 % for the group of mice treated with the RNA preparation and 67 % for those treated with B-190. In the course of analytical studies of the total yeast RNA preparation, it was found that the preparation is a mixture of single-stranded and double-stranded RNA. It was shown that only double-stranded RNA has radioprotective properties. Injection of 160 μg double-stranded RNA protects 100 % of the experimental animals from an absolutely lethal dose of gamma radiation, 9.4 Gy. It was established that the radioprotective effect of double-stranded RNA does not depend on sequence, but depends on its double-stranded form and the presence of “open” ends of the molecule. It is supposed that the radioprotective effect of double-stranded RNA is associated with the participation of RNA molecules in the correct repair of radiation-damaged chromatin in blood stem cells. The hematopoietic pluripotent cells that have survived migrate to the periphery, reach the spleen and actively proliferate. The newly formed cell population restores the hematopoietic and immune systems, which determines the survival of lethally irradiated animals

Modified siRNA effectively silence inducible immunoproteasome subunits in NSO cells

© 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.The pathogenesis of autoimmune and neurodegenerative diseases involves overexpression of inducible subunits of the immunoproteasome. However, the clinical application of inhibitors to inducible subunits of the immunoproteasome has been limited due to systemic toxicity. Here, we designed siRNAs that efficiently silence LMP2, LMP7 and MECL-1 gene expression. Inducible subunits of the immunoproteasome are complex siRNA targets because they have a long half-life; therefore, we introduced 2′-O-methyl modifications into nuclease-sensitive sites. This led to 90-95% silencing efficiency and prolonged silencing, eliminating the need for multiple transfections. Furthermore, we showed that in the absence of transfection reagent, siRNAs with lipophilic residues were able to penetrate cells more effectively and decrease the expression of inducible immunoproteasome subunits by 35% after 5 days. These results show that siRNA targeted to inducible immunoproteasome subunits have great potential for the development of novel therapeutics for autoimmune and neurodegenerative diseases

Modified siRNA effectively silence inducible immunoproteasome subunits in NSO cells

© 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.The pathogenesis of autoimmune and neurodegenerative diseases involves overexpression of inducible subunits of the immunoproteasome. However, the clinical application of inhibitors to inducible subunits of the immunoproteasome has been limited due to systemic toxicity. Here, we designed siRNAs that efficiently silence LMP2, LMP7 and MECL-1 gene expression. Inducible subunits of the immunoproteasome are complex siRNA targets because they have a long half-life; therefore, we introduced 2′-O-methyl modifications into nuclease-sensitive sites. This led to 90-95% silencing efficiency and prolonged silencing, eliminating the need for multiple transfections. Furthermore, we showed that in the absence of transfection reagent, siRNAs with lipophilic residues were able to penetrate cells more effectively and decrease the expression of inducible immunoproteasome subunits by 35% after 5 days. These results show that siRNA targeted to inducible immunoproteasome subunits have great potential for the development of novel therapeutics for autoimmune and neurodegenerative diseases

Modified siRNA effectively silence inducible immunoproteasome subunits in NSO cells

© 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.The pathogenesis of autoimmune and neurodegenerative diseases involves overexpression of inducible subunits of the immunoproteasome. However, the clinical application of inhibitors to inducible subunits of the immunoproteasome has been limited due to systemic toxicity. Here, we designed siRNAs that efficiently silence LMP2, LMP7 and MECL-1 gene expression. Inducible subunits of the immunoproteasome are complex siRNA targets because they have a long half-life; therefore, we introduced 2′-O-methyl modifications into nuclease-sensitive sites. This led to 90-95% silencing efficiency and prolonged silencing, eliminating the need for multiple transfections. Furthermore, we showed that in the absence of transfection reagent, siRNAs with lipophilic residues were able to penetrate cells more effectively and decrease the expression of inducible immunoproteasome subunits by 35% after 5 days. These results show that siRNA targeted to inducible immunoproteasome subunits have great potential for the development of novel therapeutics for autoimmune and neurodegenerative diseases