Tool For Gene Silencing Research Articles

Engineered Artificial MicroRNA Precursors Facilitate Cloning and Gene Silencing in Arabidopsis and Rice.

Plant genome sequences are presently deciphered at a staggering speed, due to the rapid advancement of high-throughput sequencing technologies. However, functional genomics significantly lag behind due to technical obstacles related to functional redundancy and mutant lethality. Artificial microRNA (amiRNA) technology is a specific, reversible, and multiplex gene silencing tool that has been frequently used in generating constitutive or conditional mutants for gene functional interrogation. The routine approach to construct amiRNA precursors involves multiple polymerase chain reactions (PCRs) that can increase both time and labor expenses, as well as the chance to introduce sequence errors. Here, we report a simplified method to clone and express amiRNAs in Arabidopsis and rice based on the engineered Arabidopsis miR319a or rice miR528 precursor, which harbor restriction sites to facilitate one-step cloning of a single PCR product. Stem-loop reverse-transcriptase quantitative PCR (RT-qPCR) and functional assays validated that amiRNAs can be accurately processed from these modified precursors and work efficiently in plant protoplasts. In addition, Arabidopsis transgenic plants overexpressing the modified miR319a precursor or its derived amiRNA could exhibit strong gene silencing phenotypes, as expected. The simplified amiRNA cloning strategy will be broadly useful for functional genomic studies in Arabidopsis and rice, and maybe other dicotyledon and monocotyledon species as well.

Insights into siRNA Transfection in Suspension: Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel.

Small interfering RNAs (siRNAs) are powerful tools for post-transcriptional gene silencing, which offers enormous opportunities for tissue engineering applications. However, poor serum stability, inefficient intracellular delivery, and inevitable toxicity of transfection reagents are the key barriers for their clinical translation. Thus, innovative strategies that allow safe and efficient intracellular delivery of the nucleic acid drugs at the desired site is urgently needed for a smooth clinical translation of therapeutically appealing siRNA-based technology. In this regard, we have developed an innovative siRNA transfection protocol that employs a short incubation time of just 5 min. This allows easy transfection in suspension followed by transplantation of the cells in a hyaluronic acid (HA) hydrogel system. We also report here the unique ability of siRNA to bind HA that was quantified by siRNA release and rheological characterization of the HA-hydrogel. Such interactions also showed promising results to deliver functional siRNA in suspension transfection conditions within 30 min using native HA, although removal of excess HA by centrifugation seem to be essential. In the 2D experiments, suspension transfection of hMSCs with RNAiMAX resulted in ≈90% gene silencing (with or without removal of the excess reagent by centrifugation), while HA demonstrated a modest ≈40% gene silencing after removal of excess reagent after 30 min. Transplantation of such transfected cells in the HA-hydrogel system demonstrated an improved knockdown (≈90% and ≈60% with RNAiMAX and HA respectively after 48 h), with lower cytotoxicity (up to 5-days) as determined by PrestoBlue assay. The gene silencing efficiency in the 2D and 3D conditions were also confirmed at the protein levels by Western blot analysis. We postulate this novel transfection method could be applied for in vivo applications as it allows minimal manipulation of cells that are to be transplanted and reduce toxicity.

Initial evidence of functional siRNA machinery in dinoflagellates

Dinoflagellates are a major group of protists widely distributed in the aquatic environments. Many species in this lineage are able to form harmful algal blooms (HAB), some even producing toxins, making this phylum the most important contributors of HAB in the marine ecosystem. Despite the ecological importance, the molecular mechanisms underpinning the basic biology and HAB formation of dinoflagellates are poorly understood. While the high-throughput sequencing studies have documented a large and growing number of genes in dinoflagellates, their functions remained to be experimentally proven using a functional genetic tool. Unfortunately, no such tool is yet available. This study was aimed to adopt the RNA interference (RNAi) gene-silencing tool for dinoflagellate research, and to investigate the potential effects of RNAi-based silencing of proton-pump rhodopsin and CO2-fixing enzyme Rubisco encoding genes in dinoflagellates. It was found that RNAi treatment caused a significant decrease in growth rate in both species. Compared with the non- endogenous target (GFP-siRNA) and the blank control, RNAi treatments also suppressed the expression of the target genes. These results constitute the first experimental evidence of the existence and operation of siRNA in two species of dinoflagellates, present initial evidence that dinoflagellate rhodopsins are functional as a supplemental energy acquisition mechanism, and provide technical information for future functional genetic research on dinoflagellates.

Functional analysis of a novel parasitic nematode-specific protein of Setaria digitata larvae in Culex quinquefasciatus by siRNA mediated RNA interference

BackgroundFunctional analysis of animal parasitic nematode genes is often quite challenging due to the unavailability of standardised in vitro culture conditions and lack of adequate tools to manipulate these genes. Therefore, this study was undertaken to investigate the suitability of Culex quinquefasciatus, as an in vivo culture platform for Setaria digitata larvae and RNA interference (RNAi), as a post-transcriptional gene silencing tool to study the roles of a vital gene that encodes a novel parasitic nematode-specific protein (SDNP).ResultsThe red colour fluorescence detected following RNAi injection to the thorax of C. quinquefasciatus indicated the uptake of dsRNA by S. digitata larvae. The reduction of SDNP transcripts in siRNA treated larvae compared to non-treated larvae, as determined by qPCR, indicated that the siRNA pathway is operational in S. digitata larvae. The observation of motility reductions and deformities during the development indicated the association of SDNP in larvae locomotion and development processes, respectively. The irregularities in the migration of larvae in mosquitoes and elevated survival rates of mosquitoes compared to their untreated counterparts indicated reduced parasitism of S. digitata larvae in mosquitoes upon targeted downregulation of SDNP by siRNA treatment.ConclusionSDNP plays vital roles in muscle contraction, locomotion, development processes, larval development and parasitism of S. digitata. Its ubiquitous presence in parasitic nematodes and its absence in their hosts provide a tantalising prospect of the possibility of targeting SDNP for future development of anthelmintic drugs. The susceptibility of the larval stages of S. digitata for RNAi in Culex quinquefasciatus was also demonstrated for the first time in this study.

Construction of an M1GS Ribozyme for Targeted and Rapid mRNA Cleavage; Application on the Ets-2 Oncogene.

RNase P-mediated cleavage of target RNAs has been proposed as a promising tool for gene silencing. Ets-2 proto-oncogene controls the expression of a wide variety of genes involved in cancer and immunity. Construction of a functional RNase P-based ribozyme (M1GS303) that targets Ets-2 mRNA. The accessible sites for targeting of Ets-2 mRNA were identified by footprinting analysis. M1GS303 ribozyme was constructed by cloning. The activity of the ribozyme in the presence or absence of spiramysin in E. coli cells and human cell lines was quantified by RT-PCR. The efficiency of the ribozyme in silencing the endogenous expression of Ets-2 in human cell lines was examined by RT-PCR, western blot and immunofluorescence analysis. In E. coli cells co-transformed with plasmids bearing M1GS303 and the ets-2 target gene, Ets-2 mRNA was decreased by 93% 12h after IPTG induction in the absence, and after 4h in the presence of spiramycin. Ets-2 was rapidly downregulated in the human embryonic kidney cell line HEK293 and the T-cell line Jurkat transfected with an M1GS303 plasmid; the silencing effect of M1GS303 was considerably faster when the cells were cultured with spiramycin. In Jurkat cells, Ets-2-downregulation resulted in upregulation of the expression of IL-2, IL-4 and IFN-α cytokine genes that have Ets-2 binding sites on their promoters, whereas it had no effect on the expression of the IL-10 gene that lacks Ets-2 binding sites on its promoter. M1GS303 ribozyme cleaves effectively Ets-2 mRNA in bacteria and mammalian cells, and its activity is enhanced by spiramycin. Downregulation of ets-2 gene in the T-cell line Jurkat upregulates IL-2, IL-4 and IFN-α cytokine genes. M1GS technology may be a better alternative to conventional gene-interference therapies and the delineation of the effects of gene silencing in various pathologies.

Argonaute Proteins and Mechanisms of RNA Interference in Eukaryotes and Prokaryotes

Noncoding RNAs play essential roles in genetic regulation in all organisms. In eukaryotic cells, many small noncoding RNAs act in complex with Argonaute proteins and regulate gene expression by recognizing complementary RNA targets. The complexes of Argonaute proteins with small RNAs also play a key role in silencing of mobile genetic elements and, in some cases, viruses. These processes are collectively called RNA interference. RNA interference is a powerful tool for specific gene silencing in both basic research and therapeutic applications. Argonaute proteins are also found in prokaryotic organisms. Recent studies have shown that prokaryotic Argonautes can also cleave their target nucleic acids, in particular DNA. This activity of prokaryotic Argonautes might potentially be used to edit eukaryotic genomes. However, the molecular mechanisms of small nucleic acid biogenesis and the functions of Argonaute proteins, in particular in bacteria and archaea, remain largely unknown. Here we briefly review available data on the RNA interference processes and Argonaute proteins in eukaryotes and prokaryotes.

Polypurine Reverse Hoogsteen Hairpins as a Gene Silencing Tool for Cancer.

Polypurine reverse Hoogsteen (PPRH) molecules are DNA hairpins formed by two polypurine strands running in an antiparallel orientation and containing no nucleotide modifications. The two strands, linked by a pentathymidine loop, are bound through intramolecular reverse Hoogsteen bonds. Then, PPRHs can bind by Watson-Crick bonds to their corresponding polypyrimidine target in the dsDNA provoking a displacement of the polypurine strand of the duplex. We described the effect and mechanisms of action of PPRHs in cells using PPRHs designed against the template and coding strands of the dhfr gene. The proof of principle of PPRHs as a therapeutic tool was established using a PPRH against survivin in a xenograft prostate cancer tumor model. To improve the PPRHs effect, the influence of the length was studied obtaining a higher efficiency with longer molecules. To decrease the possible offtarget effect, when a purine interruption is found in the pyrimidine target, the PPRH sequence should contain both strands of the complementary base opposite to the interruption. Furthermore, the stability of PPRHs is higher than that of siRNAs, as evidenced by the longer halflife of the former in different types of serum and in PC3 cells. PPRHs do not induce the levels of the transcription factors nor the proinflammatory cytokines involved in the Toll-like Receptor pathway and they do not trigger the formation of the inflammasome complex. PPRHs can be used as therapeutic tools to target genes related to cancer progression, resistance to drugs or immunotherapy approaches.

Analysis of the Role of the Drought-Induced Gene DRI15 and Salinity-Induced Gene SI1 in Alternanthera philoxeroides Plasticity Using a Virus-Based Gene Silencing Tool.

Alternanthera philoxeroides is a notoriously invasive weed that can readily adapt to different environmental conditions. Control of this weed is difficult, and it spreads easily and causes damage to native habitats and agriculture. In this study, our goal was to investigate the molecular mechanisms that lead to the ability of A. philoxeroides to invade new habitats, to adapt to environmental stresses, and to cause damage. We developed a simple and highly effective potato virus X-based virus-induced gene silencing (VIGS) approach. The VIGS approach was first used to silence the phytoene desaturase gene, which resulted in the expected photo-bleaching phenotype. Next, the VIGS approach was used to silence two additional genes, drought-induced protein gene 15 (ApDRI15) and salinity-induced protein gene 1 (ApSI1). When ApDRI15 was knocked down, the plants were more sensitive to drought stress than the control plants, with smaller leaves, shorter internodes, and lower biomass. The ApDRI15-silenced plants had lower relative water content, lower free proline levels, and higher water loss rates than the control. Silencing of ApSI1 significantly decreased tolerance to salinity, and the ApSI1-silenced plants were withered and smaller. These results indicate that the pgR107 VIGS approach is a simple and highly effective tool for dissecting gene function in A. philoxeroides. Further experiments with the VIGS approach will enhance our understanding of the molecular mechanisms of the adaptability and plasticity of A. philoxeroides and improve our ability to combat the damage caused by this weed.

Improvement of abiotic stress adaptive traits in mulberry (Morus spp.): an update on biotechnological interventions.

Mulberry (Morus spp.), being an economically important tree, is cultivated in China, India, Thailand, Brazil, Uzbekistan and other Countries across the globe, for its leaves to feed monophagous mulberry silkworm (Bombyx mori). The sustainability of silk industry is directly correlated with the production and continuous supply of high-quality mulberry leaves. In India, it is cultivated on large scale in tropical, sub-tropical and temperate regions under irrigated conditions for silkworm rearing. Drought, low temperature, high salinity and alkalinity, being experienced in widespread areas, are the major abiotic stresses, causing reduction in its potential foliage yield and quality. Further, climate change effects may worsen the productivity of mulberry in near future, not only in India but also across the globe. Although traditional breeding methods contributed immensely towards the development of abiotic stress-tolerant mulberry varieties, still there is lot of scope for implementation of modern genomic and molecular biology tools for accelerating mulberry genetic improvement programmes. This review discusses omics approaches, molecular breeding, plant tissue culture and genetic engineering techniques exploited for mulberry genetic improvement for abiotic stress tolerance. However, high-throughput biotechnological tools such as RNA interference, virus-induced gene silencing, epigenomics and genome editing tools need to be utilized in mulberry to accelerate the progress of functional genomics. The application of genomic tools such as genetic engineering, marker-assisted selection and genomic selection in breeding programmes can hasten the development of climate resilient and productive mulberry varieties leading to the vertical and horizontal expansion for quality silk production.

Gold Nanoparticle Approach to the Selective Delivery of Gene Silencing in Cancer-The Case for Combined Delivery?

Gene therapy arises as a great promise for cancer therapeutics due to its potential to silence genes involved in tumor development. In fact, there are some pivotal gene drivers that suffer critical alterations leading to cell transformation and ultimately to tumor growth. In this vein, gene silencing has been proposed as an active tool to selectively silence these molecular triggers of cancer, thus improving treatment. However, naked nucleic acid (DNA/RNA) sequences are reported to have a short lifetime in the body, promptly degraded by circulating enzymes, which in turn speed up elimination and decrease the therapeutic potential of these drugs. The use of nanoparticles for the effective delivery of these silencers to the specific target locations has allowed researchers to overcome this issue. Particularly, gold nanoparticles (AuNPs) have been used as attractive vehicles for the target-specific delivery of gene-silencing moieties, alone or in combination with other drugs. We shall discuss current trends in AuNP-based delivery of gene-silencing tools, considering the promising road ahead without overlooking existing concerns for their translation to clinics.

RNA interference as a gene silencing tool to control Tuta absoluta in tomato (Solanum lycopersicum).

RNA interference (RNAi), a gene-silencing mechanism that involves providing double-stranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer (Tuta absoluta), a major threat to commercial tomato production, can be targeted by RNAi. We selected two target genes (Vacuolar ATPase-A and Arginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet for T. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on “in planta-induced transient gene silencing” (PITGS), a well-established method for silencing plant genes, used here for the first time to deliver in planta-transcribed dsRNA to target insect genes. Tuta absoluta larvae that fed on leaves containing dsRNA of the target genes showed an ∼60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic ‘Micro-Tom’ tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage by T. absoluta in these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.

Optimization of phage λ promoter strength for synthetic small regulatory RNA-based metabolic engineering

Synthetic small regulatory RNAs (sRNAs) are gene-silencing tools that can be used to tune gene expression in prokaryotes. A recent study by our group proposed rational design principles, introduced a regulatory system that may be used to implement synthetic sRNAs, and showed their utility in metabolic engineering. The regulatory system employed the strong phage λ PR promoter to tightly control synthetic sRNA production. Here, we fine-tuned the strength of the PR promoter via mutagenesis in order to optimize the level of synthetic sRNAs while maintaining the ability of the promoter to be regulated by CI proteins. Five mutant promoters of different strengths, ranging from 24 to 87% of that of the wild-type PR promoter, were identified and confirmed to be repressed by CI proteins. A mutated promoter with only 40% of the original strength still produced enough synthetic sRNAs to inhibit the translation of the target mRNA to ~10% of the original level. As a practical application, we tested our promoters as drivers for a synthetic anti-murE sRNA, which was used to adjust the production of cadaverine. As the promoter strength decreased, the cadaverine titer first increased and then dropped. A mutated promoter with 39% of the original strength achieved the improved cadaverine titer of 2.15 g/L. The mutant promoters developed in this study should prove useful for tuning the expression levels of synthetic sRNAs for metabolic engineering.

Synthesis of an artificial Vitis vinifera miRNA 319e using overlapping long primers and its application for gene silencing

The conserved mechanism of action of micro-RNAs (miRNAs) as regulators of gene expression has allowed the use of artificial miRNAs (amiRNAs) as a powerful tool for candidate gene evaluation in plants. Based on the use of a Vitis vinifera miRNA molecule (i.e., vvi-miR319e), the present work presents a new methodology for designing artificial miR319e precursors (pre-amiR319e). As a proof of concept, we silenced the green fluorescent protein (GFP) gene in transgenic Nicotiana benthamiana plants. This methodology includes a two-step PCR reaction in which overlapping long primers allow for the complete generation of pre-amiR319e-GFP molecules that are adequate for recombination into Gateway vectors with no further requirements. The seed region in amiRNA was directed against the 3′-end portion of the GFP gene. Three groups of transformed N. benthamiana plants were generated: GFP-, amiR319e-GFP-, and GFP plus miR319e-GFP-expressing vectors. A similar group of wild-type plants was included. Confocal microscopy evaluation of these groups revealed strong silencing of the GFP phenotype in the double GFP plus amiR319e-GFP group. The molecular characterization of silenced plants was achieved via modified 5′RACE of the GFP mRNA and revealed the occurrence of a partial, 3′-end GFP mRNA molecule that was generated in planta. In addition, large-scale small RNA sequencing confirmed the occurrence of the expected 21-nt miR319e-GFP species and other 22- and 24-nt species that exhibited sequence relationships with the expected amiRNA. These results highlight the possibility of using vvi-MIR319 as a template for the generation of single amiRNAs as a tool for gene silencing in plants.

Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System.

Assessing the cell-type expression pattern of a certain gene can be achieved by using cell-type-specific gene manipulation. Recently, cre-recombinase-dependent gene-silencing tool, pSico has become popular in neuroscientific research. However, pSico has a critical limitation that gene-silenced cell cannot be identified by fluorescence, due to an excision of the reporter gene for green fluorescence protein (GFP). To overcome this limitation, we newly developed pSico-Red, with mCherry gene as a reporter outside two loxP sites, so that red mCherry signal is detected in all transfected cells. When a cell expresses cre, GFP is excised and shRNA is enabled, resulting in disappearance of GFP. This feature of pSico-Red provides not only cell-type-specific gene-silencing but also identification of cre expressing cells. Using this system, we demonstrated for the first time the neuronal expression of the Bestrophin-1 (Best1) in thalamic reticular nucleus (TRN) and TRN-neuron-specific gene-silencing of Best1. We combined adeno-associated virus (AAV) carrying Best1-shRNA in pSico-Red vector and transgenic mouse expressing cre under the promoter of distal-less homeobox 5/6 (DLX5/6), a marker for inhibitory neurons. Firstly, we found that almost all of inhibitory neurons in TRN express Best1 by immunohistochemistry. Using pSico-Red virus, we found that 80% of infected TRN neurons were DLX5/6-cre positive but parvalbumin negative. Finally, we found that Best1 in DLX5/6-cre positive neurons were significantly reduced by Best1-shRNA. Our study demonstrates that TRN neurons strongly express Best1 and that pSico-Red is a valuable tool for cell-type-specific gene manipulation and identification of specific cell population.

Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference.

RNA interference (RNAi) is an effective gene-silencing tool, and double stranded RNA (dsRNA) is considered a powerful strategy for gene function studies in insects. In the present study, we aimed to investigate the function of trehalase (TRE) genes (TRE 1-1, TRE 1-2, and TRE-2) isolated from the brown planthopper Nilaparvata lugens, a typical piercing-sucking insect in rice, and investigate their regulating roles in chitin synthesis by injecting larvae with dsRNA. The results showed that TRE1 and TRE2 had compensatory function, and the expression of each increased when the other was silenced. The total rate of insects with phenotypic deformities ranged from 19.83 to 24.36% after dsTRE injection, whereas the mortality rate ranged from 14.16 to 31.78%. The mRNA levels of genes involved in the chitin metabolism pathway in RNA-Seq and DGEP, namely hexokinase (HK), glucose-6-phosphate isomerase (G6PI) and chitinase (Cht), decreased significantly at 72 h after single dsTREs injection, whereas two transcripts of chitin synthase (CHS) genes decreased at 72 h after dsTRE1-1 and dsTREs injection. These results demonstrated that TRE silencing could affect the regulation of chitin biosynthesis and degradation, causing moulting deformities. Therefore, expression inhibitors of TREs might be effective tools for the control of planthoppers in rice.

Small interfering RNA targeting S100A4 sensitizes non-small-cell lung cancer cells (A549) to radiation treatment.

ObjectiveThis study aimed to investigate the impact of S100A4-small interfering RNA (S100A4-siRNA) on apoptosis and enhanced radiosensitivity in non-small-cell lung cancer (A549) cells. We also explored the mechanisms of radiosensitization and identified a new target to enhance radiosensitivity and gene therapy for non-small-cell lung cancer.MethodsRNA interference is a powerful tool for gene silencing. In this study, we constructed an effective siRNA to knock down S100A4. A549 cells were randomly divided into three groups: blank, negative control, and S100A4-siRNA. To investigate the effect of S100A4-siRNA, the expression of S100A4, E-cadherin, and p53 proteins and their messenger RNA (mRNA) was detected by Western blot and quantitative real-time polymerase chain reaction. Transwell chambers were used to assess cell invasion. Cell cycle and apoptosis were analyzed by flow cytometry. Radiosensitivity was determined by colony formation ability.ResultsOur results demonstrate that S100A4-siRNA effectively silenced the S100A4 gene. When siRNA against S100A4 was used, S100A4 protein expression was downregulated, whereas the expressions of E-cadherin and p53 were upregulated. In addition, a clear reduction in S100A4 mRNA levels was noted compared with the blank and negative control groups, whereas E-cadherin and p53 mRNA levels increased. Transfection with S100A4-siRNA significantly reduced the invasiveness of A549 cells. S100A4 silencing induced immediate G2/M arrest in cell cycle studies and increased apoptosis rates in A549 cells. In clonogenic assays, we used a multitarget, single-hit model to detect radiosensitivity after S100A4 knockdown. All parameters (D0, Dq, α, β) indicated that the downregulation of S100A4 enhanced radiosensitivity in A549 cells. Furthermore, S100A4-siRNA upregulated p53 expression, suggesting that S100A4 may promote A549 cell proliferation, invasion, and metastasis by regulating the expression of other proteins. Therefore, siRNA-directed S100A4 knockdown may represent a viable clinical therapy for lung cancer.ConclusionS100A4 downregulation potentially enhances the sensitivity of human A549 cells to radiotherapy.

Recombinant pre-miR-29b for Alzheimer´s disease therapeutics.

MicroRNAs are arising as the next generation of diagnostic and therapeutic tools for gene silencing. Studies demonstrated that the miR-29 expression is decreased in Alzheimer’s disease (AD) patients displaying high levels of human β-secretase (hBACE1). Recent advances toward an effective therapy for AD intend to employ miR-29 to suppress hBACE1 expression and subsequent Amyloid-β (Aβ) peptide. However, delivery of mature miRNA has demonstrated modest efficacy in vitro; therefore, the preparation of highly pure and biologically active pre-miRNA arises as one of the most important challenges in the development of these therapeutic strategies. Recently, we described a new strategy based arginine-affinity chromatography to specifically purify the recombinant pre-miR-29b. Following this strategy, the purified pre-miR-29b was successfully encapsulated into polyplexes that were further delivered in cytoplasm. It was verified that Chitosan/pre-miR-29b and Polyethylenimine/pre-miR-29b systems efficiently delivered pre-miR-29b to N2a695 cells, thus reducing the hBACE1 protein expression (around 78% and 86%, respectively) and Aβ42 levels (approximately 44% and 47%, respectively). Furthermore, pre-miR-29b downregulates the hBACE1 mRNA expression in 80%. Overall, it was demonstrated that the recombinant pre-miR-29b using polyplexes allowed to decrease the hBACE1 and Aβ42 expression levels, improving the currently available methodologies of miRNA-based therapeutics.

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.

Silencing Myostatin Using Cholesterol-conjugated siRNAs Induces Muscle Growth.

Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85–95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes.

УЧАСТЬ ПРОТЕЇНКІНАЗИ С У МЕХАНІЗМАХ ПОРУШЕННЯ СУДИННОГО ТОНУСУ ЗА УМОВ ЦУКРОВОГО ДІАБЕТУ. ЧАСТИНА 4

Diabetes mellitus (DM) is acompaining by vascular tone desorders development. Regulatory enzyme protein kinase C (PKC) is involved in mechanisms of these desorders development. Numerous studies have demonstrated that contractile responces of vascular smooth muscle are enchansed in DM and endothelium-independent PKC-mediated mechanisms are involved in this process. Such mechanisms are PKC-mediated inhibition of Ca2+ activated K+ channels (BKCa) in vascular smooth muscle cells (SMCs) and SMCs myophilaments Ca2+ sensitization. PKC is a potential therapeutic target for treating vascular diabetic complications. A few compounds among PKC inhibitors already exist, such as ruboxistaurin, indolylmaleimide and its derivatives. Recently discovered method of RNA-interference (RNAi) is an essential gene-silencing tool and can also be used for PKC inhibition and DMassociated vascular complicaions elimination.