Specific Inhibition Of Gene Expression Research Articles

Quadruplex-forming oligonucleotide targeted to the VEGF promoter inhibits growth of non-small cell lung cancer cells.

BackgroundVascular endothelial growth factor (VEGF) is commonly overexpressed in a variety of tumor types including lung cancer. As a key regulator of angiogenesis, it promotes tumor survival, growth, and metastasis through the activation of the downstream protein kinase B (AKT) and extracellular signal-regulated kinase (ERK 1/2) activation. The VEGF promoter contains a 36 bp guanine-rich sequence (VEGFq) which is capable of forming quadruplex (four-stranded) DNA. This sequence has been implicated in the down-regulation of both basal and inducible VEGF expression and represents an ideal target for inhibition of VEGF expression.ResultsOur experiments demonstrate sequence-specific interaction between a G-rich quadruplex-forming oligonucleotide encoding a portion of the VEGFq sequence and its double stranded target sequence, suggesting that this G-rich oligonucleotide binds specifically to its complementary C-rich sequence in the genomic VEGF promoter by strand invasion. We show that treatment of A549 non-small lung cancer cells (NSCLC) with this oligonucleotide results in decreased VEGF expression and growth inhibition. The VEGFq oligonucleotide inhibits proliferation and invasion by decreasing VEGF mRNA/protein expression and subsequent ERK 1/2 and AKT activation. Furthermore, the VEGFq oligonucleotide is abundantly taken into cells, localized in the cytoplasm/nucleus, inherently stable in serum and intracellularly, and has no effect on non-transformed cells. Suppression of VEGF expression induces cytoplasmic accumulation of autophagic vacuoles and increased expression of LC3B, suggesting that VEGFq may induce autophagic cell death.ConclusionOur data strongly suggest that the G-rich VEGFq oligonucleotide binds specifically to the C-rich strand of the genomic VEGF promoter, via strand invasion, stabilizing the quadruplex structure formed by the genomic G-rich sequence, resulting in transcriptional inhibition. Strand invading oligonucleotides represent a new approach to specifically inhibit VEGF expression that avoids many of the problems which have plagued the therapeutic use of oligonucleotides. This is a novel approach to specific inhibition of gene expression.

Small Interfering RNA–Mediated Suppression of Fas Modulate Apoptosis and Proliferation in Rat Intervertebral Disc Cells

Study DesignIn vitro cell culture model.PurposeTo investigate the effect of small interfering RNA (siRNA) on Fas expression, apoptosis, and proliferation in serum-deprived rat disc cells.Overview of LiteratureSynthetic siRNA can trigger an RNA interference (RNAi) response in mammalian cells and precipitate the inhibition of specific gene expression. However, the potential utility of siRNA technology in downregulation of specific genes associated with disc cell apoptosis remains unclear.MethodsRat disc cells were isolated and cultured in the presence of either 10% fetal bovine serum (FBS) (normal control) or 0% FBS (serum deprivation to induce apoptosis) for 48 hours. Fas expression, apoptosis, and proliferation were determined. Additionally, siRNA oligonucleotides against Fas (Fas siRNA) were transfected into rat disc cells to suppress Fas expression. Changes in Fas expression were assessed by reverse transcription-polymerase chain reaction and semiquantitatively analyzed using densitometry. The effect of Fas siRNA on apoptosis and proliferation of rat disc cells were also determined. Negative siRNA and transfection agent alone (Mock) were used as controls.ResultsSerum deprivation increased apoptosis by 40.3% (p<0.001), decreased proliferation by 45.3% (p<0.001), and upregulated Fas expression. Additionally, Fas siRNA suppressed Fas expression in serum-deprived cultures, with 68.5% reduction at the mRNA level compared to the control cultures (p<0.001). Finally, Fas siRNA–mediated suppression of Fas expression significantly inhibited apoptosis by 9.3% and increased proliferation by 21% in serum-deprived cultures (p<0.05 for both).ConclusionsThe observed dual positive effect of Fas siRNA might be a powerful therapeutic approach for disc degeneration by suppression of harmful gene expression.

Lipopolyplexes comprising imidazole/imidazolium lipophosphoramidate, histidinylated polyethyleneimine and siRNA as efficient formulation for siRNA transfection

Lipopolyplexes formulations resulting from association of nucleic acid, cationic liposomes and a cationic polymer are attracting formulations for siRNA delivery. Herein, imidazole- and imidazolium-based liposomes in association with histidinylated polymers are studied to produce siRNA lipopoplyplexes (LPRi) subsequently used for gene silencing. Several kinds of imidazole/histidine liposomes and cationic polymers are tested. The gene silencing effect is evaluated with synthetic siRNA directed against EGFP or luciferase mRNA, in HeLa cells stably expressing EGFP or B16F10 melanoma cells stably expressing luciferase, respectively. SiRNA formulations are compared with those prepared using some commercial transfection reagents. One formulation called His-lPEI LPRi100 comprising siRNA, histidinylated lPEI (His-lPEI) and liposomes 100 made with O,O-dioleyl-N-[3N-(N-methylimidazolium iodide)propylene] phosphoramidate and O,O-dioleyl-N-histamine phosphoramidate appears to give the best specific inhibition of gene expression at 10nM siRNA in a dose-dependent manner with low cytotoxicity. This formulation exhibits a size and a zeta potential of 60 nm and +84 mV, respectively. According to our previous works, histidinylated lipopolyplexes appears as a versatile formulation for DNA, mRNA and siRNA transfection.

Dichloromethane extracts of Sophora japonica L. stimulate osteoblast differentiation in mesenchymal stem cells

Sophora japonica L. fruit prevents bone loss by inhibiting osteoclast activity. We hypothesized that S japonica L. extracts could promote osteoblast differentiation. To test this hypothesis, we investigated the effect of S japonica L. on osteoblast differentiation and identified the bioactive compound(s) from S japonica L. The mature fruit of S japonica L. was partitioned with ethanol, hexane, dichloromethane (DCM), ethyl acetate, and butanol, and their effects were tested on osteoblast differentiation of C3H10T1/2 cells. DCM fractionated extracts were identified as the most osteogenic fractions. DCM fractionated extracts dose-dependently stimulated alkaline phosphatase activity and matrix mineralization. The DCM fractions also induced expression of osteoblast markers such as alkaline phosphatase, osterix, and osteocalcin in C3H10T1/2 and primary bone marrow cells. Genistein was found abundantly in the DCM fractions. Furthermore, the genistein and DCM fractions similarly modulated the expression of estrogen target genes and were both active in transfection assays that measured estrogen agonistic activity. Finally, pharmacological inhibition by treatment with an estrogen receptor antagonist or specific inhibition of gene expression by small interference RNAs targeted to estrogen receptor-β abolished the effects of the DCM extracts, further supporting the idea that the genistein in the DCM extracts mediated the pro-osteogenic effects. Taken together, we identified genistein as the key phytoestrogen responsible for the effects of S japonica L. on osteoblast differentiation.

Influence of the Internalization Pathway on the Efficacy of siRNA Delivery by Cationic Fluorescent Nanodiamonds in the Ewing Sarcoma Cell Model

Small interfering RNAs (siRNAs) are powerful tools commonly used for the specific inhibition of gene expression. However, vectorization is required to facilitate cell penetration and to prevent siRNA degradation by nucleases. We have shown that diamond nanocrystals coated with cationic polymer can be used to carry siRNAs into Ewing sarcoma cells, in which they remain traceable over long periods, due to their intrinsic stable fluorescence. We tested two cationic polymers, polyallylamine and polyethylenimine. The release of siRNA, accompanied by Ewing sarcoma EWS-Fli1 oncogene silencing, was observed only with polyethylenimine. We investigated cell penetration and found that the underlying mechanisms accounted for these differences in behavior. Using drugs selectively inhibiting particular pathways and a combination of fluorescence and electronic microscopy, we showed that siRNA gene silencing occurred only if the siRNA:cationic nanodiamond complex followed the macropinocytosis route. These results have potential implications for the design of efficient drug-delivery vectors.

AAV-mediated in vivo knockdown of luciferase using combinatorial RNAi and U1i

RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i). U1i is a gene-silencing technique that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined effect allows usage of minimal doses, thereby avoiding toxicity while retaining high target inhibition. As a proof of concept, we investigated knockdown of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs showed additive reduction of luciferase expression up to 95% in vitro. We attained similar knockdown when RNAi and U1i constructs were hydrodynamically transfected into murine liver, demonstrating for the first time successful in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knockdown in vivo.

The advent of RNA interference in Entomology

RNA interference (RNAi) is a cellular process by which an mRNA is targeted for degradation by a small interfering RNA that contains a strand complementary to a fragment of the target mRNA, resulting in sequence specific inhibition of gene expression. The discovery of RNAi enabled the use of loss-of-function analyses in many non-model insects other than Drosophila to elucidate the roles of specific genes. The RNAi approach has been widely used on insects in several fields, including embryogenesis, pattern formation, reproduction, biosynthesis and behavior. The increasing availability of insect genomes has made the RNAi technique an indispensable technique for characterizing gene functions in insects. Here we review the current status of RNAi-based experiments in insects and the applications of RNAi for species-specific insecticides, focusing on non-drosophilid insects. We also identify future applications for RNAi-based studies in Entomology.

Combination of RNA interference and U1 inhibition leads to increased inhibition of gene expression

RNA interference (RNAi) has been revolutionary for the specific inhibition of gene expression. However, the application of RNAi has been hampered by the fact that many siRNAs induce dose-dependent unwanted secondary effects. Therefore, new methods to increase inhibition of gene expression with low doses of inhibitors are required. We have tested the combination of RNAi and U1i (U1 small nuclear RNA—snRNA—interference). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to target a pre-mRNA and inhibit its gene expression by blocking nuclear polyadenylation. The combination of RNAi and U1i resulted in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. The increased inhibition observed is stable over time and allows higher inhibition than the best obtained with either of the inhibitors alone even with decreased doses of the inhibitors. We believe that the combination of RNAi and U1i will be of interest when higher inhibition is required or when potent inhibitors are not available. Also, the combination of these techniques would allow functional inhibition with a decreased dose of inhibitors, avoiding toxicity due to dose-dependent unwanted effects.

In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA.

BackgroundSelf-complementary RNA transcripts form a double-stranded RNA (dsRNA) that triggers a sequence-specific mRNA degradation, in a process known as RNA interference (RNAi), leading to gene silencing. In vascular plants, RNAi molecules trafficking occur between cells and systemically throughout the plant. RNAi signals can spread systemically throughout a plant, even across graft junctions from transgenic to non-transgenic stocks. There is also a great interest in applying RNAi to pathogenic fungi. Specific inhibition of gene expression by RNAi has been shown to be suitable for a multitude of phytopathogenic filamentous fungi. However, double-stranded (ds)RNA/small interfering (si)RNA silencing effect has not been observed in vivo.ResultsThis study demonstrates for the first time the in vivo interference phenomenon in the pathogenic fungus Fusarium verticillioides, in which expression of an individual fungal transgene was specifically abolished by inoculating mycelial cells in transgenic tobacco plants engineered to express siRNAs from a dsRNA corresponding to the particular transgene.ConclusionThe results provide a powerful tool for further studies on molecular plant-microbe and symbiotic interactions. From a biotechnological perspective, silencing of fungal genes by generating siRNAs in the host provides a novel strategy for the development of broad fungi-resistance strategies in plants and other organisms.

Evaluation and application of a luciferase fusion system for rapidin vivoanalysis of RNAi targets and constructs in plants

The practical use of RNA-mediated approaches including antisense RNA, ribozymes and siRNAs for specific inhibition of gene expression is limited by lack of simple quantitative methods to rapidly test efficacy in vivo. There have been indications that cotransfer of target::reporter gene fusions with constructs designed against the target sequence, followed by quantification of transient reporter gene activity might be effective. Here, we report detailed testing of the approach in plants, using diverse target::luciferase fusions and antisense or ribozyme constructs. We used quantitative transient luciferase activity (Luc) assays to test antisense constructs against beta-glucuronidase, PR glucanase, vacuolar invertase and cucumber mosaic virus, as well as ribozymes against watermelon mosaic virus and tobacco anionic peroxidase. For constructs previously tested in transgenic plants, the results correspond well with those from the transient expression assay. Target susceptibility was generally not strongly influenced by luciferase fusion, and the assay was not highly dependent on target sequence length. Some sequences reduced Luc activity below the level for reliable quantification, but suitable alternative fusions were readily produced. Transcriptional and translation fusions were effective for 5' target::luc constructs. Translational fusions were more reliable for luc::target 3' constructs. With minimal preliminary work to prepare suitable target::luciferase fusions, the approach appears generally applicable for rapid in vivo validation of effectiveness and specificity of constructs designed for RNA-mediated down-regulation of plant genes.

RNAi‐based therapeutics–current status, challenges and prospects

RNA interference (RNAi) is a collection of small RNA directed mechanisms that result in sequence specific inhibition of gene expression. The notion that RNAi could lead to a new class of therapeutics caught the attention of many investigators soon after its discovery. The field of applied RNAi therapeutics has moved very quickly from lab to bedside. The RNAi approach has been widely used for drug development and several phase I and II clinical trials are under way. However, there are still some concerns and challenges to overcome for therapeutic applications. These include the potential for off-target effects, triggering innate immune responses and most importantly obtaining specific delivery into the cytoplasm of target cells. This review focuses on the current status of RNAi-based therapeutics, the challenges it faces and how to overcome them.

Highly Active Low Magnesium Hammerhead Ribozyme

Hammerhead (HH) ribozymes can be used for highly specific inhibition of gene expression through the degradation of target mRNA. In vitro experiments with minimal HH domains demonstrated that the efficiency of catalysis is highly dependent on concentration of magnesium ions. Optimal ion requirements for HH-catalysed RNA cleavage are far from these found in the cell. Recently, it has been proposed that the efficiency of HH ribozymes can be increased at low magnesium concentration through stabilization of a catalytically active conformation by tertiary interactions between helices I and II. We designed a ribozyme stabilized by GAAA tetraloop and its receptor motifs and demonstrated that it can efficiently catalyse target RNA hydrolysis at submillimolar Mg(2+) concentrations in vitro as well as in cultured cells. Both unmodified and locked nucleic acid-modified extended ribozymes proved superior to the minimal core ribozyme and DNAzyme against the same target sequence.

Overcoming the classical multidrug resistance phenotype by adenoviral delivery of anti-MDR1 short hairpin RNAs and ribozymes

Simultaneous resistance of cancer cells to multiple cytotoxic drugs, multidrug resistance (MDR), is the major limitation to the successful chemotherapeutic treatment of disseminated neoplasms. The 'classical' MDR phenotype is conferred by MDR1/P-glycoprotein (MDR1/P-gp) that is expressed in almost 50% of human cancers. Recent developments in the use of small interfering RNAs for specific inhibition of gene expression have highlighted their potential use as therapeutic agents. DNA cassettes encoding RNA polymerase III promoter-driven siRNA-like short hairpin RNAs (shRNAs) allow long-term expression of therapeutic RNAs in targeted cells. A variety of viral vectors have been used to deliver such cassettes to mammalian cells. In this study, the construction of different adenoviruses for anti-MDR1/P-gp shRNA delivery in different human multidrug-resistant cancer cells was investigated. The efficiency of the shRNAs was compared to adenoviral delivery of an anti-MDR1/P-gp ribozyme construct. It could be demonstrated that MDR1/P-gp mRNA and protein expression could be completely inhibited by adenoviral delivery of anti-MDR1/P-gp shRNAs. This downregulation in mRNA and protein expression was accompanied by a complete inhibition of the pump activity of MDR1/P-gp and a reversal of the multidrug-resistant phenotype. By application of adenoviral encoded anti-MDR1/P-gp ribozyme construct merely weak effects on gene expression were observed. In conclusion, the data demonstrate that adenoviral delivery of shRNAs can chemosensitize human cancer cells, that adenoviral delivery of shRNAs is much more effective than adenoviral delivery of ribozymes, and that adenovirus-based vectors can be very effective agents for efficient delivery of therapeutic RNA molecules.

E3-Independent Monoubiquitination of Ubiquitin-Binding Proteins

Ubiquitin (Ub)-binding domains (UBDs) are key elements in conveying Ub-based cellular signals. UBD-containing proteins interact with ubiquitinated targets and control numerous biological processes. They themselves undergo UBD-dependent monoubiquitination, which promotes intramolecular binding of the UBD to the attached Ub and leads to their inactivation. Here, we report that, in contrast to the established ubiquitination pathway, the presence of UBDs allows the ubiquitination of host proteins independently of E3 ligases. UBDs of different types, including UBA, UIM, UBM, NFZ, and UBZ, can directly cooperate with Ub-charged E2 enzymes to promote monoubiquitination. Using FRET and siRNA technologies, we verify that Ub-loaded E2 and substrates interact in cells and that E2 enzymes are essential for their monoubiquitination in vivo. This modification is mechanistically and functionally distinct from E3-mediated and growth factor-dependent monoubiquitination.

Gene Silencing of Cyclooxygenase-2 mRNA by RNA Interference in Bovine Cumulus-Granulosa Cells

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.

Antisense DNA and RNA: Potential Therapeutics for Viral Infection

Antisense DNA and RNA are valuable tools to inhibit expression of a target gene in a sequence-specific manner. These molecules are not only widely used for gene functional study but also for therapeutic purpose. The strategy for therapeutics is attributed to its specific inhibition of gene expression of pathogens or disease-causing genes. Three types of anti-mRNA strategies can be distinguished, including antisense oligodeoxynucleotide (AODN), nucleic acid enzymes, and double-stranded small interfering RNA (siRNA). In this article we overview the basic principles of AODN and siRNA and then focus on their potential applications in antiviral therapy including our own data on coxsackieviral infection, a common pathogen of human myocarditis. In addition, we also briefly discuss the problems and difficulties in these drug developments, which need to be overcome to achieve the final goal in clinical application.

Alu-linked hairpins efficiently mediate RNA interference with less toxicity than do H1-expressed short hairpin RNAs

RNA interference has become a powerful tool for specific inhibition of gene expression in mammalian cells. Expression constructs allow for the long-term delivery of short interfering RNAs, usually through the expression of Pol III-transcribed hairpins. In some instances, these expression systems have been shown to have side effects, including induction of the interferon response and cytotoxicity. Here we demonstrate that H1-expressed hairpins, as well as the cloning vector, reduce the plating efficiency of HeLa cells. This toxicity is abrogated by coexpression of the hairpin in the same transcript as a human Alu repetitive element. These Alu-linked hairpins retain the ability to knock down expression of target mRNAs. This modification, which we term SINE (short interspersed repetitive element)-enhanced short hairpin RNA, provides an alternative expression system for hairpins with reduced side effects.

Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes

RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21–25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA.

Adenovirus-Mediated Silencing of Huntingtin Expression by shRNA

Huntington's disease (HD) is an inherited autosomal dominant, neurodegenerative disease that is caused by a gain of function mutation characterized by the expansion of a CAG trinucleotide repeat in exon 1 of the huntingtin (htt) gene. Since hairpin small interference RNA (shRNA) technology allows inhibition of specific gene expression in vitro and in vivo, vector-mediated expression of an shRNA directed to htt mRNA could form the basis of a new treatment modality for HD. By initial plasmid transfection of 293 cells, we identified one exon 1-targeted shRNA, which efficiently inhibited expression of an htt exon 1-GFP fusion protein and the endogenous htt gene. A replication-deficient adenovirus (Ad) vector Adie-1-1 was constructed to express this shRNA from the U6 promoter. In A549 cells expressing exon 1 of htt with an expanded CAG allele, Adie- 1-1 efficiently prevented htt exon 1 expression and htt aggregate formation. In addition, in different neuronal and nonneuronal cell lines, Adie-1-1 efficiently inhibited the expression of endogenous htt. Together, this data indicates the delineation of an shRNA strategy that may become the basis for treatment of HD.

The post-transcriptional gene silencing pathway in Eucalyptus

Post-transcriptional gene silencing (PTGS) is a conserved surveillance mechanism that identifies and cleaves double-stranded RNA molecules and their cellular cognate transcripts. The RNA silencing response is actually used as a powerful technique (named RNA interference) for potent and specific inhibition of gene expression in several organisms. To identify gene products in Eucalyptus sharing similarities with enzymes involved in the PTGS pathway, we queried the expressed sequence tag database of the Brazilian Eucalyptus Genome Sequence Project Consortium (FORESTs) with the amino acid sequences of known PTGS-related proteins. Among twenty-six prospected genes, our search detected fifteen assembled sequences encoding products presenting high level of similarity (E value < 10-40) to proteins involved in PTGS in plants and other organisms. We conclude that most of the genes known to be involved in the PTGS pathway are represented in the FORESTs database.