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At least 181 records · Page 10Linked to original sources
PubMed2007

[Effect of ST6Gal I siRNA-mediated gene silencing on the adhesion and invasion of SW480 cells].

AIM: To study the effect of synthesized ST6Gal I specific siRNA on the adhesion and invasiveness of human colon carcinoma cell line SW480 with over expression of ST6Gal I. METHODS: A double strand small interference RNA (siRNA) targeting ST6Gal I was designed and synthesized, and then transfected into SW480 cells by lipofectmine 2000. SW480 cells were cultured and divided into 4 groups: blank control group, liposome control group, non-specific siRNA group and ST6Gal I siRNA group. The expression of ST6Gal I mRNA was examined by RT-PCR and the amount of alpha-2, 6-sialylation on the SW480 cell surface was detected by flow cytometry. The adhesion and invasion of SW480 cells to extracellular matrix (ECM) were analyzed by using CytoMatrix kit and cell invasion assay kit, respectively. RESULTS: After SW480 cells were transfected for 48 hours, the expression of ST6Gal I mRNA in ST6Gal I siRNA group was significantly decreased compared with that in the blank control group, liposome control group, and non-specific siRNA group (P<0.05). After SW480 cells were transfected for 72 hours, the amount of alpha-2, 6-sialylation on cell surface, the adhesion and invasion of the cells in ST6Gal I siRNA group were markedly lower than those in the other 3 groups (P<0.05). CONCLUSION: The chemically synthesized specific siRNA targeting ST6Gal I can effectively inhibit the expression of ST6Gal I and reduce cell adhesion and invasion to ECM in SW480 cells. Our research is important for further study of anti-tumor treatment with RNA interference.

Cell Adhesion↗
PubMed2004

Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells.

BACKGROUND: RNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector-based siRNA-expression systems that can induce RNAi in living cells. METHODS: We describe here a comparative analysis of various siRNA-expression systems, in which we examined the effects of stem length, loop sequence and insertion of mutation(s) and/or bulges in the stem sequence on silencing effects and on the stability of the vectors. RESULTS: As a result of the comparative analysis, we determined the following optimized siRNA-expression system: U6 promoter-driven hairpin-type dsRNA with 21-nt stem length, three to four mutations in the sense strand only, and the optimized 9-nt loop sequence, derived from microRNA. Moreover, we demonstrate that the siRNA-expression system with a tetracycline-regulated U6 promoter(s) could have the potential to control RNAi in cells, and that the HIV vector-mediated transfer of an siRNA-expression cassette into cells resulted in efficient silencing of a target gene at a multiplicity of infection as low as five. CONCLUSION: The mutated hairpin siRNAs and their genetically stable coding vectors could be very useful for gene knockdown experiments, and could further benefit gene therapy using RNAi.

Base Sequence↗
PubMed2006

Screening of siRNA target sequences by using fragmentized DNA.

BACKGROUND: RNA interference (RNAi) has become a powerful tool in silencing target genes in various organisms. In mammals, RNAi can be induced by using short interfering RNA (siRNA). The efficacy of inducing RNAi in mammalian cells by using siRNA depends very much on the selection of the target sequences. METHODS: We developed an siRNA target sequence selection system by first constructing parallel-type siRNA expression vector libraries carrying siRNA expression fragments originating from fragmentized target genes, and then using a group selection system. For a model system, we constructed parallel-type siRNA expression vector libraries against DsRed and GFP reporter genes. RESULTS: We carried out the first screening of groups containing more than 100 random siRNA expression plasmids in total for each target gene, and successfully obtained target sequences with very strong efficacy. Furthermore, we also obtained some clones that express dsRNAs of various lengths that might induce cytotoxicity. CONCLUSIONS: This system should allow us to perform screening for powerful target sequences, by including all possible target sequences for any gene, even without knowing the whole sequence of the target gene in advance. At the same time, target sequences that should be avoided due to cytotoxicity can be identified.

Base Sequence↗
PubMed2006

Targeting of gene expression by siRNA in CML primary cells.

Development of array methods contributes to elucidation of many genes expressed during oncogenesis. Our array-based analyses of gene expression in patients with chronic myeloid leukemia (CML) revealed several genes (MMP8, MMP9, PCNA, JNK2, MAPK p38) with significant increased expression. We suppose that the genes may be implicated in the disease development and a siRNA-suppression can elucidate their functions in leukemogenesis. One of the crucial requirements for this purpose is a high efficiency of siRNA delivery into CML primary cells. Using fluorescein-labeled siRNAs we systematically tested a variety of physical and chemical non-vector based transfection methods in order to evaluate which of them gave the most suitable transfer. Chemically synthesized siRNAs against mentioned genes were transfected into the cells and level of knockdown was determined by real time RT-PCR. Chemical transfection reagents (Oligofectamine, Metafectene, siPORT Amine) commonly used to transfect siRNAs in CML cell lines showed very low siRNA delivery in CML primary cells-mRNA levels decreased at the most to 76%. Electroporation achieved better results (suppression to 63%) but it was associated with high degree of cell death (more than 60%). In the study we obtained the best transfection efficiency using nucleofector technology. Gene expressions ranged 22-37% that remained from original levels. According to our results, nucleofection appears to be the only suitable non-viral method for siRNA delivery into the hard-to-transfect CML primary cells.

Adult↗
PubMed2006

Biophysical and structural characterization of polyethylenimine-mediated siRNA delivery in vitro.

PURPOSE: The goals of this study were as follows: 1) to evaluate the efficacy of different polyethylenimine (PEI) structures for siRNA delivery in a model system, and 2) to determine the biophysical and structural characteristics of PEI that relate to siRNA delivery. MATERIALS AND METHODS: Biophysical characterization (effective diameter and zeta potential), cytotoxicities, relative binding affinities and in vitro transfection efficiencies were determined using nano-complexes formed from PEI's of 800, 25,000, (both branched) and 22,000 (linear) molecular weights at varying N:P ratios and siRNA concentrations. The HR5-CL11 cell line stably expressing luciferase was used as a model system in vitro. RESULTS: Successful siRNA delivery was observed within a very narrow window of conditions, and only with the 25,000 branched PEI at an N:P ratio of 6:1 and 8:1 and with 200 nM siRNA. While the zeta potential and size of PEI:siRNA complexes correlated to transfection efficacy in some cases, complex stability may also affect transfection efficacy. CONCLUSIONS: The ability of PEI to transfer functionally active siRNA to cells in culture is surprisingly dependent on its biophysical and structural characteristics when compared to its relative success and ease of use for DNA delivery.

Biophysical Phenomena↗
PubMed2006

Strand-specific silencing of a picornavirus by RNA interference: evidence for the superiority of plus-strand specific siRNAs.

RNA interference triggered by small interfering RNAs (siRNAs) can be used to effectively contain viral spread. Here, we report on the mechanism of action of siRNAs targeting the medically important coxsackievirus B3 (CVB-3) as a typical representative of viruses with a non-segmented RNA genome in positive-strand orientation. Antiviral siRNAs can be designed to target the genomic (+)-strand, the (-)-strand that occurs as a replication intermediate, or both. In the present study, two complementary and systematic approaches are presented providing direct evidence that silencing of the viral (+)-strand is the key to inhibit CVB-3: first, we used rational siRNA design to direct silencing activity specifically against either of the two viral strands. As a second approach, we employed siRNA containing modified nucleotides to render them specific for one of the virus RNAs. Experiments with infectious coxsackievirus revealed that the inhibitory efficiency correlates exclusively with the activity of the siRNAs directed against the viral (+)-strand. Our finding that only (+)-strand specific siRNAs exert significant antiviral potency may hold true for other RNA viruses with (+)-stranded genomes as well and may therefore be helpful in the development of efficient strategies to inhibit virus propagation.

Animals↗
PubMed2004

Transfection of "naked" siRNA results in endosomal uptake and metabolic impairment in cultured neurons.

RNA interference is rapidly becoming a powerful tool for gene silencing in mammalian cells. Introduction of siRNA into primary cells, however, remains one of the major difficulties of this novel technique. Using cationic lipid-based transfection reagents satisfactory transfection results are observed in cell lines, but low transfection efficiency and cytotoxicity limit applications in primary cells, especially primary neurons. The application of "naked" siRNA has been previously used successfully in nematodes and mammals in vivo. We therefore evaluated the effects of non-cationic-lipid-based siRNA application to primary hippocampal neuron cultures. "Naked" siRNA was bound to the cell surface and was taken up into endosomes. No significant silencing effect of endogenous or reporter genes was observed, rather application of "naked" siRNA was accompanied by a moderate downregulation of metabolic activity in culture. We postulate that endosomal degradation of "naked" siRNA in neurons prevents the induction of significant RNAi-mediated mRNA-downregulation and is accompanied by a global impairment of the cell metabolism. Transfection methods circumventing the endosomal pathway therefore might prove useful for siRNA transduction of primary neurons.

Animals↗
PubMed2004

Many commonly used siRNAs risk off-target activity.

Using small interfering RNA (siRNA) to induce sequence specific gene silencing is fast becoming a standard tool in functional genomics. As siRNAs in some cases tolerate mismatches with the mRNA target, knockdown of genes other than the intended target could make results difficult to interpret. In an investigation of 359 published siRNA sequences, we have found that about 75% of them have a risk of eliciting non-specific effects. A possible cause for this is the popular BLAST search engine, which is inappropriate for such short oligos as siRNAs. Furthermore, we used new special purpose hardware to do a transcriptome-wide screening of all possible siRNAs, and show that many unique siRNAs exist per target even if several mismatches are allowed. Hence, we argue that the risk of off-target effects is unnecessary and should be avoided in future siRNA design.

Base Sequence↗
PubMed2005

Synthesis of novel siRNAs having thymidine dimers consisting of a carbamate or a urea linkage at their 3' overhang regions and their ability to suppress human RNase L protein expression.

In order to examine the effect of modifications at the 3' overhang regions of short interfering RNAs (siRNAs) on their gene-silencing activities, we designed and synthesized novel siRNAs having thymidine dimers consisting of a carbamate or a urea linkage at their 3' overhang regions. Suppression of human RNase L protein expression by these siRNAs was analyzed by immunoblot with RNase L-specific antibody. It was found that, at 24 h post-transfection, the modified siRNAs having the thymidine dimers with the carbamate and urea linkage suppress the protein expression 78 and 37 times more efficiently than that with the natural phosphodiester linkage, respectively. Furthermore, the siRNA containing the carbamate linkage was 37 times more resistant to nucleolytic degradation by snake venom phosphodiesterase than the siRNA consisting of the natural phosphodiester linkage. Thus, the RNA duplexes having the thymidine dimers with the carbamate or urea linkage at their 3' overhang regions will be promising candidates for novel siRNA molecules to down-regulate protein expression.

Carbamates↗
PubMed2005

Prediction of siRNA knockdown efficiency using artificial neural network models.

Selective knockdown of gene expression by short interference RNAs (siRNAs) has allowed rapid validation of gene functions and made possible a high throughput, genome scale approach to interrogate gene function. However, randomly designed siRNAs display different knockdown efficiencies of target genes. Hence, various prediction algorithms based on siRNA functionality have recently been constructed to increase the likelihood of selecting effective siRNAs, thereby reducing the experimental cost. Toward this end, we have trained three Back-propagation and Bayesian neural network models, previously not used in this context, to predict the knockdown efficiencies of 180 experimentally verified siRNAs on their corresponding target genes. Using our input coding based primarily on RNA structure thermodynamic parameters and cross-validation method, we showed that our neural network models outperformed most other methods and are comparable to the best predicting algorithm thus far published. Furthermore, our neural network models correctly classified 74% of all siRNAs into different efficiency categories; with a correlation coefficient of 0.43 and receiver operating characteristic curve score of 0.78, thus highlighting the potential utility of this method to complement other existing siRNA classification and prediction schemes.

Algorithms↗
PubMed2006

Inflammatory cytokine induction by siRNAs is cell type- and transfection reagent-specific.

Specific knock-down of cellular gene expression using short, interfering RNAs (siRNAs) has become a powerful tool for functional genomics studies and a promising future therapeutic approach. However, recent studies have revealed that siRNAs can trigger an innate immune response upon intravenous administration in mice and transfection into purified immune cells by upregulating inflammatory cytokine levels. In this study, we demonstrate that transfection of siRNAs into several established human cancer cell lines can also induce inflammatory cytokine production regardless of the sequence of the siRNA used. The amount of inflammatory cytokine induction is cell type-specific, whereas the induction pattern is siRNA sequence-specific. We also show that, in a given cell type, different transfection reagents have different effects on inflammatory cytokine induction. Our results highlight the promiscuity of siRNA-triggered innate immune responses in human cancer cell lines and call for caution in the design and analysis of siRNA-based experiments.

Cell Line↗
PubMed2005

microRNA-directed phasing during trans-acting siRNA biogenesis in plants.

Plants and animals use small RNAs (microRNAs [miRNAs] and siRNAs) as guides for posttranscriptional and epigenetic regulation. In plants, miRNAs and trans-acting (ta) siRNAs form through distinct biogenesis pathways, although they both interact with target transcripts and guide cleavage. An integrated approach to identify targets of Arabidopsis thaliana miRNAs and ta-siRNAs revealed several new classes of small RNA-regulated genes, including conventional genes such as Argonaute2 and an E2-ubiquitin conjugating enzyme. Surprisingly, five ta-siRNA-generating transcripts were identified as targets of miR173 or miR390. Rather than functioning as negative regulators, miR173- and miR390-guided cleavage was shown to set the 21-nucleotide phase for ta-siRNA precursor processing. These data support a model in which miRNA-guided formation of a 5' or 3' terminus within pre-ta-siRNA transcripts, followed by RDR6-dependent formation of dsRNA and Dicer-like processing, yields phased ta-siRNAs that negatively regulate other genes.

Arabidopsis↗
PubMed2005

Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis.

In higher eukaryotes, miRNAs and siRNAs guide translational inhibition, mRNA cleavage, or chromatin regulation. We found that the antisense overlapping gene pair of Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH), a stress-related gene, and SRO5, a gene of unknown function, generates two types of siRNAs. When both transcripts are present, a 24-nt siRNA is formed by a biogenesis pathway dependent on DCL2, RDR6, SGS3, and NRPD1A. Initial cleavage of the P5CDH transcript guided by the 24-nt siRNA establishes a phase for the subsequent generation of 21-nt siRNAs by DCL1 and further cleavage of P5CDH transcripts. The expression of SRO5 is induced by salt, and this induction is required to initiate siRNA formation. Our data suggest that the P5CDH and SRO5 proteins are also functionally related, and that the P5CDH-SRO5 gene pair defines a mode of siRNA function and biogenesis that may be applied to other natural cis-antisense gene pairs in eukaryotic genomes.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed2006

Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis.

MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) in plants form through distinct pathways, although they function as negative regulators of mRNA targets by similar mechanisms . Three ta-siRNA gene families (TAS1, TAS2, and TAS3) are known in Arabidopsis thaliana. Biogenesis of TAS3 ta-siRNAs, which target mRNAs encoding several AUXIN RESPONSE FACTORs (including ARF3/ETTIN and ARF4 ) involves miR390-guided processing of primary transcripts, conversion of a precursor to dsRNA through RNA-DEPENDENT RNA POLYMERASE6 (RDR6) activity, and sequential DICER-LIKE4 (DCL4)-mediated cleavage events. We show that the juvenile-to-adult phase transition is normally suppressed by TAS3 ta-siRNAs, in an ARGONAUTE7-dependent manner, through negative regulation of ARF3 mRNA. Expression of a nontargeted ARF3 mutant (ARF3mut) in a wild-type background reproduced the phase-change phenotypes detected in rdr6-15 and dcl4-2 mutants, which lose all ta-siRNAs. Expression of either ARF3 or ARF3mut in rdr6-15 plants, in which both endogenous and transgenic copies of ARF3 were derepressed, resulted in further acceleration of phase change and severe morphological and patterning defects of leaves and floral organs. In light of the functions of ARF3 and ARF4 in organ asymmetry, these data reveal multiple roles for TAS3 ta-siRNA-mediated regulation of ARF genes in developmental timing and patterning.

Arabidopsis↗
PubMed2006

DSTHO: database of siRNAs targeted at human oncogenes: a statistical analysis.

Existing treatments of human cancer, which is characterized by abnormal proliferation of cells often lead to fatal outcomes. Sequence selective silencing of oncogene expression using siRNA technology is emerging as a potential solution for cancer treatment. The exclusive selectivity and easy application to virtually any therapeutic target including intracellular factors and transcription factors renders siRNA oligonucleotide applications very promising. However, synthesis of siRNA having sufficient knockdown efficiency is laborious and cost intensive. The database is designed in order to aid the synthesis of siRNAs, which target human oncogenes (OsiRNAs). It provides OsiRNAs of known efficacy from previous experiments with links to published literature and theoretically pre-generated putative target sequences. In addition, links to available theoretical tools, databases and literature corresponding to siRNAs in general are also provided. The links to literature provide information about role of siRNA in therapeutics, chemical properties and transfection methods. Statistical analysis of mono-, di- and tri- mers located in OsiRNAs of known efficacies is performed to identify positional preferences and screen specific motifs. This analysis aids the design and synthesis of effective siRNAs, which particularly target human oncogenes. The database can be accessed at .

Base Composition↗
PubMed2006

Knockdown of mouse VCAM-1 by vector-based siRNA.

Graft rejection is critically dependent on the recruitment of leukocytes via adhesion molecules on the endothelium, and inhibition of these interactions can prolong graft survival. We have therefore developed an approach using siRNA to inhibit the expression of VCAM-1 in endothelial cells. We transfected siRNA constructs into murine corneal and vascular endothelium and looked at expression of VCAM-1 and other surface molecules by flow cytometry. Adhesion assays (both static and under flow) were used to determine the effect of VCAM-1 inhibition. The activation of cellular stress responses was assessed by RT-PCR. Constructs encoding siRNA can block expression of VCAM-1 in both corneal and vascular endothelial cells (in the latter case after cytokine stimulation). Inhibition of VCAM-1 expression reduced the ability of T cells to adhere to endothelium. However, there were non-specific effects of siRNA expression, including upregulation of (Programmed Death Ligand 1) PDL1 and decreased cell growth. Analysis of stress pathways showed that the endothelial cells transfected with siRNA had upregulated molecules associated with cell stress. While these data are supportive of a potential therapeutic role for siRNA constructs in blocking the expression of adhesion molecules, they also highlight potential non-specific effects of siRNA that must be carefully considered in any application of this technology.

Animals↗
PubMed2006

Insulin signaling in vascular endothelial cells: a key role for heterotrimeric G proteins revealed by siRNA-mediated Gbeta1 knockdown.

Activation of insulin receptors stimulates the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in vascular endothelial cells. Heterotrimeric G proteins appear to modulate some of the cellular responses that are initiated by receptor tyrosine kinases, but the roles of specific G protein subunits in signaling are less clearly defined. We found that insulin treatment of cultured bovine aortic endothelial cells (BAEC) activates the alpha isoform of PI3-K (PI3-Kalpha) and discovered that purified G protein Gbeta1gamma2 inhibits PI3-Kalpha enzyme activity. Transfection of BAEC with a duplex siRNA targeting bovine Gbeta1 leads to a 90% knockdown in Gbeta1 protein levels, with no effect on expression of other G protein subunits. siRNA-mediated Gbeta1 knockdown markedly and specifically potentiates insulin-dependent activation of kinase Akt, likely reflecting the removal of the inhibitory effect of Gbetagamma on PI3-Kalpha activity. Insulin-induced tyrosine phosphorylation of insulin receptors is unaffected by Gbeta1 siRNA. By contrast, Gbeta1 knockdown leads to a significant decrease in the level of serine phosphorylation of the insulin receptor substrate IRS-1. We explored the effects of siRNA on several serine/threonine protein kinases that have been implicated in insulin signaling. Gbeta1 siRNA significantly attenuates phosphorylation of the 70 kDa ribosomal protein S6 kinase (p70S6K) in the basal state and following insulin treatment. We also found that IGF-1-initiated activation of Akt is significantly enhanced after siRNA-mediated Gbeta1 knockdown, while IGF-1-induced p70S6K activation is markedly suppressed following transfection of Gbeta1 siRNA. We propose that Gbeta1 participates in the activation of p70S6K, which in turn promotes the serine phosphorylation and inhibition of IRS-1. Taken together, these studies suggest that Gbeta1 plays an important role in insulin and IGF-1 signaling in endothelial cells, both by inhibiting the activity of PI3-Kalpha and by stimulating pathways that lead to activation of protein kinase p70S6K and to the serine phosphorylation of IRS-1.

Animals↗
PubMed2005

Design of a genome-wide siRNA library using an artificial neural network.

The largest gene knock-down experiments performed to date have used multiple short interfering/short hairpin (si/sh)RNAs per gene. To overcome this burden for design of a genome-wide siRNA library, we used the Stuttgart Neural Net Simulator to train algorithms on a data set of 2,182 randomly selected siRNAs targeted to 34 mRNA species, assayed through a high-throughput fluorescent reporter gene system. The algorithm, (BIOPREDsi), reliably predicted activity of 249 siRNAs of an independent test set (Pearson coefficient r = 0.66) and siRNAs targeting endogenous genes at mRNA and protein levels. Neural networks trained on a complementary 21-nucleotide (nt) guide sequence were superior to those trained on a 19-nt sequence. BIOPREDsi was used in the design of a genome-wide siRNA collection with two potent siRNAs per gene. When this collection of 50,000 siRNAs was used to identify genes involved in the cellular response to hypoxia, two of the most potent hits were the key hypoxia transcription factors HIF1A and ARNT.

Algorithms↗