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At least 91 records · Page 5Linked to original sources
PubMed2006

Inhibition of connective tissue growth factor by siRNA prevents liver fibrosis in rats.

BACKGROUND: Connective tissue growth factor (CTGF) is a highly profibrogenic molecule implicated in hepatic fibrogenesis. Small interfering RNA (siRNA) is an effective tool to silence gene expression post-transcriptionally. Therefore, we conducted an investigation to determine if intraportal vein siRNA injection targeting CTGF inhibits CTGF expression on rat liver in vivo and furthermore whether it protects the liver from liver fibrosis. METHODS: Some rats received carbon tetrachloride (CCl4) by subcutaneous injections every three days for six consecutive weeks, and meantime they also obtained either siRNA (0.1 mg/kg) targeting CTGF, saline or a control siRNA by intraportal vein injection to rats' liver at the same pattern. Other rats received CCl4 by subcutaneous injection for 2 weeks, followed by CCl4 and CTGF siRNA intraportal vein injection for four more weeks. RESULTS: Intraportal vein injection of CTGF siRNA specifically reduced the expression of CTGF protein in rat liver, and these effects were maintained for 3 days. Six weeks after CCl4 injection, prominent upregulations were observed in the gene expressions of CTGF, type I, III collagen, laminin, tissue inhibitor metal proteinase-1 (TIMP-1) and transforming growth factor-beta1 (TGF-beta1) in saline or control siRNA-treated rats livers. Administrating CTGF siRNA for 4 or 6 weeks, by contrast, markedly attenuated the induction of CTGF, type I, III collagen, laminin, TIMP-1 and TGF-beta1 genes, whereas Smad2, 7 gene expression was not affected. The number of active hepatic stellate cells (HSCs) determined by the expression of alpha-smooth muscle actin was also significantly decreased. The CTGF siRNA treatment markedly reduced serum procollagen type III, hepatic hydroxyproline and liver fibrosis staging. CONCLUSIONS: Silencing CTGF expression with siRNA demonstrates therapeutic potential to prevent liver fibrosis by inhibiting HSC activation with consequent extracellular matrix accumulation and the upregulation of TGF-beta1 gene expression.

Animals↗
PubMed2006

Effects of siRNAs in combination with Gleevec on K-562 cell proliferation and Bcr-Abl expression.

RNA interference (RNAi) is the repression of gene expression through a cellular mechanism of transcript-specific mRNA degradation. RNAi has been observed in human cells and applied to the modulation of a variety of human transcripts. Our goals were to deliver small interfering RNA (siRNA) using a liposome-based method, and to show Bcr-Abl siRNA specificity against K-562 cells, alone or in combination with Gleevec. Both synthetic (syn) siRNA, consisting of homogeneous 21-nucleotide-long RNA duplexes specific for the Bcr-Abl fusion site, and recombinant (r)-generated Bcr-Abl siRNA were employed. siRNA was transfected into K-562 cells with greater than 90% efficiency using RNAiFect, as judged by fluorescence analysis. The Bcr-Abl transcript was inhibited using either siRNA preparation as measured by RT-PCR or real-time PCR. The IC(50) of Gleevec in the K-562 subline F(1) was lowered over 3-fold from 0.2 to 0.06 muM in cells transfected with either syn or rBcr-Abl siRNA. No effect was observed in cells after transfection with an irrelevant control siRNA. Therefore, K-562 cells transfected with RNAifect deliver Bcr-Abl siRNA efficiently and the Bcr-Abl siRNA decreased the IC(50) of Gleevec required to inhibit the high levels of Bcr-Abl protein found in K-562 cells.

Base Sequence↗
PubMed2006

DRB4-dependent TAS3 trans-acting siRNAs control leaf morphology through AGO7.

trans-acting siRNAs (ta-siRNAs) are endogenous RNAs that direct the cleavage of complementary mRNA targets . TAS gene transcripts are cleaved by miRNAs; the cleavage products are protected against degradation by SGS3, copied into dsRNA by RDR6, and diced into ta-siRNAs by DCL4 . We describe hypomorphic rdr6 and sgs3 Arabidopsis mutants, which do not exhibit the leaf developmental defects observed in null mutants and which, like null alleles, are impaired in sense-transgene-induced posttranscriptional gene silencing and virus resistance. Null rdr6 and sgs3 mutants lack TAS1, TAS2, and TAS3 ta-siRNAs and overaccumulate ARF3/ETTIN and ARF4 mRNAs, which are TAS3 ta-siRNA targets. A hypomorphic rdr6 mutant accumulates wild-type TAS3 ta-siRNA levels but not TAS1 and TAS2 ta-siRNAs, suggesting that TAS3 is required for proper leaf development. Consistently, tas3 but not tas1 or tas2 mutants exhibits leaf morphology defects, and ago7/zip and drb4 mutants, which exhibit leaf morphology defects, lack TAS3 but not TAS1 and TAS2 ta-siRNAs in leaves. These results indicate that the dsRNA binding protein DRB4 is required for proper ta-siRNA production, presumably by interacting with DCL4, an interaction analogous to that of HYL1 with DCL1 during miRNA production , and that TAS3 ta-siRNAs are required for proper leaf development through the action of AGO7/ZIPPY.

Alleles↗
PubMed2003

Analysis of the 3(')-hydroxyl group in Drosophila siRNA function.

Members of the RNA-dependent RNA polymerase (RdRP) gene family have been shown to be essential for dsRNA-mediated gene silencing based on genetic screens in a variety of organisms, including Caenorhabditis elegans, Arabidopsis, Neurospora, and Dictyostelium. A hallmark of this process is the formation of small 21- to 25-bp dsRNAs, termed siRNAs for small interfering RNAs, which are derived from the dsRNA that initiates gene silencing. We have developed methods to demonstrate that these siRNAs produced in Drosophila embryo extract can be uniformly incorporated into dsRNA in a template-specific manner that is subsequently degraded by RNase III-related enzyme activity to create a second generation of siRNAs. SiRNA function in dsRNA synthesis and mRNA degradation depends upon the integrity of the 3'-hydroxyl of the siRNA, consistent with the interpretation that siRNAs serve as primers for RdRP activity in the formation of dsRNA. This process of siRNA incorporation into dsRNA followed by degradation and the formation of new siRNAs has been termed "degradative PCR" and the proposed mechanism is consistent with the genetic and biochemical data derived from studies in C. elegans, Arabidopsis, Drosophila, and Dictyostelium. The methods used to study the function of both natural and synthetic siRNAs in RNA interference in Drosophila embryo extracts are detailed. The importance of the 3'-hydroxyl group for siRNA function and its incorporation into dsRNA is emphasized and the results support a model that places RNA-dependent RNA polymerase as a key mediator in the RNA interference mechanism in Drosophila.

Animals↗
PubMed2006

In situ analysis of single-stranded and duplex siRNA integrity in living cells.

To attain the full therapeutic promise of short interfering RNA (siRNA), it is believed that improvements such as increased biostability are critical. Regrettably, thus far, insufficient in situ data are on hand regarding the intracellular stability of siRNAs. We report on the use of an advanced fluorescence-based method to probe the nucleolytic decay of double labeled siRNAs, which are subject to fluorescence resonance energy transfer (FRET). In vitro measurements with RNAse A and cellular extracts demonstrate that the ratio of acceptor (5'-Cy5) to donor (3'-rhodamine green) fluorescence can be used to study the degradation of the labeled siRNA substrates upon donor excitation. Intracellular FRET analysis showed substantial degradation of single-stranded siRNA, whereas duplex siRNA stayed intact during the measured time period. These data underline the high intrinsic nuclease resistance of unmodified duplex siRNA and prove that cellular persistence is much more critical for the single-stranded structure. For the first time, the stability of siRNA is investigated in real-time inside living cells. The fluorescence-based method presented here is a straightforward technique to gain direct information on siRNA integrity inside living cells and provides a bright outlook to learn more about the intracellular fate of siRNA therapeutics.

Animals↗
PubMedDate not supplied

Potent and specific inhibition of retrovirus production by coexpression of multiple siRNAs directed against different regions of viral genomes.

siRNA-mediated RNA degradation has been demonstrated to act as an antiviral system in many species. Here we describe inhibition of retrovirus production by multiple siRNAs designed to target various regions of the viral genomes. Using murine leukemia virus (MuLV) as a model, we demonstrate that the virus production can be inhibited by 77% in siLTR2 (a siRNA targeting the U3 region of MuLV) expression vector transfected cells. Coexpression of siLTR2 with siPsi2 (a siRNA targeting the 3' Psi (packaging signal sequence) results in 93% suppression of the virus production, suggesting that an increased inhibition of the virus production can be achieved by coexpression of multiple siRNAs to target different regions of the viral RNA simultaneously. Our results also indicate that not all sequences of the viral RNA are equally accessible to siRNA. We show that U3 region of MuLV is more accessible to siRNA, whereas the packaging signal sequence, especially the region adjacent to 5'LTR, is less accessible to siRNA, partly as a result of the binding of Gag precursors. Furthermore, we demonstrate that coexpression of siLTR2 with siPsi2 in virus producer cells leads to 88% knockdown of viral titer, showing the benefit of coexpression of multiple siRNAs for potent suppression of virus production in the setting of an established infection. Moreover, we demonstrate that infection of MuLV in cells that stably coexpress siLTR2 with siPsi2 diminishes by 77%. Taken together, we establish that siRNA-mediated gene silencing can suppress multiple steps of the retrovirus life cycle, offering a potential for both treating virus-associated diseases and preventing viral infection.

Animals↗
PubMed2005

Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1.

The high mutation rate of the human immunodeficiency virus (HIV) makes it difficult for any therapy employing a single anti-HIV targeting mechanism to sustain prolonged effect. In an attempt to explore novel therapy for AIDS, we developed and tested lentiviral small interfering RNA (siRNA) vectors targeting multiple highly conserved regions in the HIV type 1 (HIV-1) genome. The siRNA expression cassette was cloned into an extensively deleted HIV-1-derived lentiviral self-inactivating insulator (SIN) insulator [corrected] vector. Although some of the siRNAs targeting sites were also present in the helper construct of the vector system, the production of these lentiviral siRNA vectors were not significantly affected. When tested against different HIV-1 strains including pNL4-3 (subtype B), p89.6 (subtype B) and p90CF402.1.8 (subtype A/E recombinant), the siRNAs targeting conserved gag, pol, int and vpu, but not U3, nef or U5 regions, efficiently inhibited replication of all three viral strains. These lentiviral siRNA vectors also protected host cells from syncytium-forming macrophage- and T-cell-tropic HIV-1-induced cytotoxicity. Transduction of a long-term chronically infected human lymphoma cell line with lentiviral siRNAs resulted in stable inhibition of HIV-1 replication. Northern analysis showed that both genomic and subgenomic viral RNA species were downregulated. In addition, the viral RNA was inhibited in both the nuclear and cytoplasmic compartments of [corrected] chronically infected cells after prolonged passage, suggesting that [corrected] lentiviral siRNAs have a nuclear effect [corrected] Using these lentiviral siRNA [corrected] vectors, we further demonstrated reduced replication kinetics of HIV-1 in primary human peripheral blood lymphocytes. These results suggest that lentiviral siRNAs targeting multiple conserved HIV-1 sequences holds significant promise for the treatment of HIV-1 infections.

Base Sequence↗
PubMed2003

Inhibition of HBV replication by siRNA in a stable HBV-producing cell line.

Potent inhibition of endogenous gene expression by RNA interference has been achieved by using sequence-specific posttranscriptional gene silencing through the action of small interfering RNA molecules (siRNA). In these reports, the natural function of genes could be deduced through the ensuing loss of function. Based on the extraordinary effectiveness in silencing endogenous genes, we wondered whether siRNA could be applied against viral replication in a hepatitis B virus (HBV) model using HBV-specific siRNA. To test this idea, HepG2 2.2.15, a human hepatoblastoma cell line that constitutively produces infectious HBV particles, was transfected with HBV-specific siRNAs and controls. HBV surface antigen (HBsAg) secretion into culture media was inhibited by 78%, 67%, and 42% with siRNA against the polyadenylation (PA), precore (PreC), and surface (S) regions, respectively, compared with controls as detected by enzyme-linked immunosorbent assay. After exposure to HBVPA siRNA, Northern blot analysis showed that HBV pregenomic RNA levels were decreased by 72%, and levels of HBV RNA containing the polyadenylation signal sequence were suppressed by 86%, as detected by RNase protection assay. Levels of HBV core-associated DNA, a replication intermediate, also decreased by 71%. Immunocytochemistry revealed that 30% to 40% of the cells transfected with HBVPA siRNA were completely negative for detectable HBsAg levels. Controls consisting of treatment with HBV-specific siRNA alone, lipofection reagent alone, or random double-stranded RNA (dsRNA) lipofection complex failed to decrease HBV surface antigen, HBV messenger RNA (mRNA), or core-associated HBV-DNA levels. In conclusion, siRNA inhibits hepatitis B viral replication in a cell culture system. Future studies are needed to explore the specific delivery of siRNA to liver cells in vivo and the applicability of this approach.

Cell Line↗
PubMed2003

The apparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome.

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.

Base Sequence↗
PubMed2006

REM sleep changes in rats induced by siRNA-mediated orexin knockdown.

Short interfering RNAs (siRNA) targeting prepro-orexin mRNA were microinjected into the rat perifornical hypothalamus. Prepro-orexin siRNA-treated rats had a significant (59%) reduction in prepro-orexin mRNA compared to scrambled siRNA-treated rats 2 days postinjection, whereas prodynorphin mRNA was unaffected. The number of orexin-A-positive neurons on the siRNA-treated side decreased significantly (23%) as compared to the contralateral control (scrambled siRNA-treated) side. Neither the colocalized dynorphin nor the neighbouring melanin-concentrating hormone neurons were affected. The number of orexin-A-positive neurons on the siRNA-treated side did not differ from the number on the control side 4 or 6 days postinjection. Behaviourally, there was a persistent (approximately 60%) increase in the amount of time spent in rapid eye movement (REM) sleep during the dark (active) period for 4 nights postinjection, in rats treated with prepro-orexin siRNA bilaterally. This increase occurred mainly because of an increased number of REM episodes and decrease in REM-to-REM interval. Cataplexy-like episodes were also observed in some of these animals. Wakefulness and NREM sleep were unaffected. The siRNA-induced increase in REM sleep during the dark cycle reverted to control values on the 5th day postinjection. In contrast, the scrambled siRNA-treated animals only had a transient increase in REM sleep for the first postinjection night. Our results indicate that siRNA can be usefully employed in behavioural studies to complement other loss-of-function approaches. Moreover, these data suggest that the orexin system plays a role in the diurnal gating of REM sleep.

Animals↗
PubMed2004

A protein sensor for siRNA asymmetry.

To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two approximately 21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. We show that in Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2. R2D2 binds the siRNA end with the greatest double-stranded character, thereby orienting the heterodimer on the siRNA duplex. Strong R2D2 binding requires a 5'-phosphate on the siRNA strand that is excluded from the RISC. Thus, R2D2 is both a protein sensor for siRNA thermodynamic asymmetry and a licensing factor for entry of authentic siRNAs into the RNAi pathway.

Animals↗
PubMed2005

Variants of bcl-2 specific siRNA for silencing antiapoptotic bcl-2 in pancreatic cancer.

BACKGROUND AND AIMS: Pancreatic cancer remains a devastating diagnosis with only limited therapeutic options. Specific inhibition of expression of target genes has become possible using small interfering (si) RNAs. We therefore investigated how far siRNA specific for bcl-2 may serve as a therapeutic option for pancreatic cancer in vitro and in vivo. METHODS: siRNAs targeting two different regions in the bcl-2 gene were transfected to YAP C and DAN G pancreatic carcinoma cells and human foreskin fibroblasts. Permutations were generated by changing 3' and 5' overhangs and varying the length of the paired RNA duplex. Transfection efficacy was determined using FITC labelled siRNAs and fluorescence microscopy. Cell survival and apoptosis were quantified at 24-120 hours. Pancreatic cancer xenografts in male nude mice were treated intraperitoneally with siRNAs daily for 24 days. siRNA pharmacokinetics in vivo were assessed using radioactively labelled siRNAs. Total protein and RNA were extracted for western Blot analysis and quantitative polymerase chain reaction. RESULTS AND CONCLUSIONS: Bcl-2 specific siRNAs specifically inhibited expression of the target gene in vitro and in vivo. Antiproliferative and proapoptotic effects were observed in tumour cells but not in fibroblasts or non-malignant tissues. siRNA permutations and diverse overhangs influenced gene silencing efficacy. siRNA was quickly distributed to all organs and excreted via the kidney and liver. Bcl-2 specific siRNA is a promising adjunctive treatment for pancreatic carcinoma.

3' Untranslated Regions↗
PubMed2006

Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo.

BACKGROUND: The cationic lipid Genzyme lipid (GL) 67 is the current "gold-standard" for in vivo lung gene transfer. Here, we assessed, if GL67 mediated uptake of siRNAs and asODNs into airway epithelium in vivo. METHODS: Anti-lacZ and ENaC (epithelial sodium channel) siRNA and asODN were complexed to GL67 and administered to the mouse airway epithelium in vivo Transfection efficiency and efficacy were assessed using real-time RT-PCR as well as through protein expression and functional studies. In parallel in vitro experiments were carried out to select the most efficient oligonucleotides. RESULTS: In vitro, GL67 efficiently complexed asODNs and siRNAs, and both were stable in exhaled breath condensate. Importantly, during in vitro selection of functional siRNA and asODN we noted that asODNs accumulated rapidly in the nuclei of transfected cells, whereas siRNAs remained in the cytoplasm, a pattern consistent with their presumed site of action. Following in vivo lung transfection siRNAs were only visible in alveolar macrophages, whereas asODN also transfected alveolar epithelial cells, but no significant uptake into conducting airway epithelial cells was seen. SiRNAs and asODNs targeted to beta-galactosidase reduced betagal mRNA levels in the airway epithelium of K18-lacZ mice by 30% and 60%, respectively. However, this was insufficient to reduce protein expression. In an attempt to increase transfection efficiency of the airway epithelium, we increased contact time of siRNA and asODN using the in vivo mouse nose model. Although highly variable and inefficient, transfection of airway epithelium with asODN, but not siRNA, was now seen. As asODNs more effectively transfected nasal airway epithelial cells, we assessed the effect of asODN against ENaC, a potential therapeutic target in cystic fibrosis; no decrease in ENaC mRNA levels or function was detected. CONCLUSION: This study suggests that although siRNAs and asODNs can be developed to inhibit gene expression in culture systems and certain organs in vivo, barriers to nucleic acid transfer in airway epithelial cells seen with large DNA molecules may also affect the efficiency of in vivo uptake of small nucleic acid molecules.

Animals↗
PubMed2006

Therapeutic potential of siRNA-mediated transcriptional gene silencing.

RNA interference (RNAi) and specifically the use of small interfering RNAs (siRNAs) represents a potentially new paradigm in gene knockout technology. Clearly siRNAs can be used to knockdown the expression of a targeted transcript in what has been termed posttranscriptional gene silencing (PTGS). While there are a plethora of reports applying siRNA-mediated PTGS the limitation of the duration of the effect remains. Recently, in human cells, siRNAs have been shown, similar to plants and Schizosaccharomyces pombe, to mediate transcriptional gene silencing (TGS). The observation that siRNAs can function in a TGS manner in human cells suggests that, similar to plants, human genes may also be able to be silenced more permanently via epigenetic modifications. The ramifications of siRNA-mediated TGS in humans suggest that longer term suppression of gene function can be obtained via siRNA-directed chromatin modifications. Undoubtedly the potential to employ siRNA technology is broader than once envisioned in human cells and suggests that siRNA-mediated TGS is not simply limited to PTGS. The potential to utilize siRNAs to direct epigenetic changes in local chromatin structure offers a new therapeutic avenue that could prove remarkably robust and of immeasurable therapeutic value in the directed control of target gene expression.

Animals↗
PubMed2003

siRNA-mediated antitumorigenesis for drug target validation and therapeutics.

Application of siRNA to knockdown a specific gene requires target mRNA accessibility, effective intracellular delivery of siRNA into target cells and potent siRNA inhibition of target mRNA. Use of siRNA as a tool is advancing in almost every field of biomedical research, but some of the most dynamic and exciting applications of siRNA are in cancer research. This review summarizes the results obtained with siRNA in cancer, in particular functional validation of tumorigenic genes in cell culture and animal tumor models, effective siRNA delivery systems, efficiency of siRNA agents compared with antisense oligonucleotides and efforts for potential therapeutic development. Along with the rapidly growing literature on using siRNA as a functional genomic tool, there is emerging evidence that siRNA may represent a novel therapeutic modality for cancer treatment when optimized local and systemic delivery systems are available.

Animals↗
PubMed2005

Inhibition of vascular endothelial growth factor gene expression by T7-siRNAs in cultured human retinal pigment epithelial cells.

BACKGROUND: Retinal pigment epithelial (RPE) cells play an important role in the occurrence of choroidal neovascularization (CNV). Vascular endothelial growth factor (VEGF) as a positive regulatory growth factor is produced by the RPE in an autocrine or paracrine manner, promoting CNV development. Duplexes of 21 nt RNAs, known as short interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference when introduced into mammalian cells. We searched for an efficient siRNA to interfere with VEGF expression in RPE cells and shed light on the treatment of CNV. METHODS: Human primary RPE (hRPE) cells were cultured and identified. Three pairs of siRNAs were designed according to the sequence of VEGF 1-5 extrons and synthesized by T7 RNA polymerase transcription in vitro. To evaluate the inhibitory activity of T7-siRNAs, hRPE cells were transfected via siPORT Amine. The interfering effect of T7-siRNAs in hRPE cells was examined by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence. RESULTS: Three pairs of T7-siRNAs synthesized by in vitro transcription with T7 RNA polymerase suppressed VEGF gene expression with efficiency from 65% to 90%. T7-siRNA (B), targeted region at 207 nt to 228 nt and double stranded for 21 nt with 2 nt UU 3' overhangs, was the most effective sequence tested for inhibition of VEGF expression in hRPE cells. Compared with nontransfected cells, the mean fluorescence in hRPE cells transfected with T7-sRNAs was significantly less (P < 0.01). siRNA with a single-base mismatch and ssRNA(+) did not show suppressing effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect (5 to 10 pmol). CONCLUSION: T7-siRNA can effectively and specifically suppress VEGF expression in hRPE cells and may be a new way to treat CNV.

Base Sequence↗
PubMed2006

siRNA-mediated silencing of Cockayne Cyndrome group B gene potentiates radiation-induced apoptosis and antiproliferative effect in HeLa cells.

BACKGROUND: Cockayne syndrome (CS) is a rare human genetic disorder characterized by increased UV sensitivity, developmental abnormalities and premature aging. Cells isolated from individuals with CS have a defect in transcription-coupled DNA repair. Despite the repair defect, there is no any increased risk of spontaneous or UV-induced cancer for CS individuals. The strategy of RNA interfering was used here to explore the potential radiosensitizing and anticancer activity of targeting CS group B (CSB) gene. METHODS: The vectors encoding CSB-specific siRNAs were constructed by inserting duplex siRNA encoding oligonucleotides into the plasmid P(silencer TM 3.1). The cell lines expressing the CSB-siRNA were generated from HeLa cells transfected with the above vectors. Colony-forming ability was used to assay cell survival. Cell cycle was analyzed by FACScan flow cytometry. The apoptosis was measured by detecting the accumulation of sub-G(1) population as well as by fluorescence staining assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to semi-quantify mRNA expression. Protein level was detected by Western blotting analysis. RESULTS: Two constructs encoding CSB-specific siRNA were generated, both of them resulted in remarkable suppression on CSB expression in HeLa cells, and led to an increased sensitivity to (gamma-ray and UV light. siRNA-mediated silencing of CSB decreased cell proliferation rate, increased spontaneous apoptosis as well as the occurrence of UV- or cisplatin-induced apoptosis by 2 to 3.5 fold. A significant S phase blockage and a remarkable reduction of G(1) population were induced in control HeLa cells at 18 hours after being exposed to 10 J/m(2) of UV light. The S phase blockage was also observed in UV-irradiated CSB-siRNA transfected HeLa cells, but the extent of increased S phase population was lower than that in the UV-irradiated control cells. No or a relative weak reduction on G(1) phase population was observed in UV-irradiated CSB-siRNA transfected HeLa cells. In addition, siRNA-mediated silencing of CSB promoted the elimination of G(2)/M phase cells after UV light radiation. CONCLUSIONS: siRNA-mediated silencing of CSB causes cells to proliferate more slowly, sensitize cells to genotoxicants, and modify UV radiation-induced cell cycle changes. siRNA-mediated inactivation of CSB could be an attractive strategy for ameliorating cancer therapy, which can be fulfilled via the combination of gene therapy and sensitization of radiotherapy or chemotherapy.

Apoptosis↗
PubMed2005

Lipid-mediated siRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels.

BACKGROUND: Efficient in vivo vectors are needed to exploit the enormous potential of RNA interference (RNAi). Such methods require optimisation for specific delivery routes, tissues and usages. We tested the capacity of different non-viral vectors and formulation methods for inhibition of exogenous (luciferase) gene expression when used to introduce small interfering RNA (siRNA) into the mouse brain in vivo. METHODS: Polyethylenimine (PEI)-based polyplexes and JetSI (a mixture of cationic lipids)-based lipoplexes were used to vectorise plasmid DNA encoding the firefly Photinus pyralis luciferase gene and picomolar amounts of siRNA directed against this gene. Two controls were used, DNA encoding an unrelated luciferase from Renilla reniformis and a mutated siRNA sequence. RESULTS: First, we found that linear PEI, although efficient for delivering nucleic acids to cells, did not permit development of siRNA activity within the dose range tested (<0.5 pmol). Second, various combinations of cationic lipids were tried and the best formulation was found to be a combination of JetSI with the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE). Efficient inhibition of target, firefly luciferase was obtained with exceedingly low amounts of siRNA: 78 +/- 6% inhibition at 24 h post-transfection with 0.2 pmol siRNA. This inhibition was dose-dependent and specific. No effect was seen on the control gene, co-transfected Renilla luciferase, and the control mutated siRNA sequence had no effect on the targeted firefly luciferase. CONCLUSIONS: We have optimised an efficient cationic lipoplex method for delivery of siRNA into the newborn mouse brain. Specific inhibition of exogenous target gene expression is obtained with picomolar amounts of siRNA.

Analysis of Variance↗