SEARCH · PubMed Health

Results for “Small interfering RNA”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 343 records · Page 19Linked to original sources

PubMed2006

Argonaute slicing is required for heterochromatic silencing and spreading.

Small interfering RNA (siRNA) guides dimethylation of histone H3 lysine-9 (H3K9me2) via the Argonaute and RNA-dependent RNA polymerase complexes, as well as base-pairing with either RNA or DNA. We show that Argonaute requires the conserved aspartate-aspartate-histidine motif for heterochromatic silencing and for ribonuclease H-like cleavage (slicing) of target messages complementary to siRNA. In the fission yeast Schizosaccharomyces pombe, heterochromatic repeats are transcribed by polymerase II. We show that H3K9me2 spreads into silent reporter genes when they are embedded within these transcripts and that spreading requires read-through transcription, as well as slicing by Argonaute. Thus, siRNA guides histone modification by basepairing interactions with RNA.

Amino Acid Motifs↗

PubMed2006

Silencing of p29 affects DNA damage responses with UV irradiation.

Human p29 is a newly identified nuclear protein whose function is largely undetermined. We found that p29 associated with chromatin, interacted with MCM3, and localized with proliferating cell nuclear antigen foci in the S phase. Silencing of p29 using small interfering RNA duplexes reduced DNA synthesis and increased the expression of p107, a member of the RB family, and of cyclin-dependent kinase inhibitor p21, accompanied with a decreased expression of DNA polymerase alpha. Lethal events consisting of premature chromatin condensation with a reduced Chk1 phosphorylation were observed in p29-depleted cells in response to UV irradiation. Intriguingly, the phosphorylation of ataxia telangectasia-mutated kinases at S1981 was suppressed in p29-depleted HeLa cells with UV irradiation, but not in hydroxyurea- and ionizing radiation-treated cells. Taken together, these results reveal a novel function of p29 in the regulation of DNA replication checkpoint responses.

Apoptosis↗

PubMed2006

Competitive control of independent programs of tumor necrosis factor receptor-induced cell death by TRADD and RIP1.

Stimulation of tumor necrosis factor receptor 1 (TNFR1) can initiate several cellular responses, including apoptosis, which relies on caspases, necrotic cell death, which depends on receptor-interacting protein kinase 1 (RIP1), and NF-kappaB activation, which induces survival and inflammatory responses. The TNFR-associated death domain (TRADD) protein has been suggested to be a crucial signal adaptor that mediates all intracellular responses from TNFR1. However, cells with a genetic deficiency of TRADD are unavailable, precluding analysis with mature immune cell types. We circumvented this problem by silencing TRADD expression with small interfering RNA. We found that TRADD is required for TNFR1 to induce NF-kappaB activation and caspase-8-dependent apoptosis but is dispensable for TNFR1-initiated, RIP1-dependent necrosis. Our data also show that TRADD and RIP1 compete for recruitment to the TNFR1 signaling complex and the distinct programs of cell death. Thus, TNFR1-initiated intracellular signals diverge at a very proximal level by the independent association of two death domain-containing proteins, RIP1 and TRADD. These single transducers determine cell fate by triggering NF-kappaB activation, apoptosis, and nonapoptotic death signals through separate and competing signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Purified Argonaute2 and an siRNA form recombinant human RISC.

Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp-His (DDH) motif. In the RISC reconstitution system, the siRNA 5' phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.

Adenosine Triphosphate↗

PubMed2006

In vitro and in vivo evidence of metallopanstimulin-1 in gastric cancer progression and tumorigenicity.

PURPOSE: The metallopanstimulin-1 (MPS-1) gene is a growth factor-inducible gene, which is highly expressed in many human cancers and may be involved in the progression towards tumor malignancy. However, it is unclear whether MPS-1 plays any role in gastric cancer development or progression. Our studies were designed to clarify the MPS-1 expression pattern and to explore its potential role in gastric cancer. EXPERIMENTAL DESIGN: The expression pattern of MPS-1 was determined in primary gastric cancer specimens and gastric cancer cell lines via immunohistochemistry and Western blotting. To investigate the functional significance of MPS-1 expression, three small interfering RNA (siRNA) expression plasmids were constructed and transfected into gastric cancer cell line SGC7901. The stable cell lines transfected with the siRNA targeting MPS-1 mRNA plasmids were selected and the biological features of these cells were examined. RESULTS: MPS-1 was overexpressed in 86% of the gastric cancer tissues and all gastric cancer cells. In addition, MPS-1 expression was significantly increased and corresponded with the tumor-node-metastasis clinical stage, and was significantly higher in the late stage (P < 0.01). The MPS-1 expression level was significantly decreased in the transfected cells with MPS-1-specific siRNA expression plasmid pRNAT-133. Furthermore, the stable transfected cancer cells exhibited an increase in the incidence of spontaneous apoptosis and a decrease in growth ability and tumorigenicity in nude mice. CONCLUSIONS: These results provide strong evidence that MPS-1 plays an important role in gastric cancer cell proliferation and development, and suggests that MPS-1 is a promising target for gastric cancer treatment.

Animals↗

PubMed2004

A novel eIF5A complex functions as a regulator of p53 and p53-dependent apoptosis.

Although eukaryotic translation initiation factor 5A (eIF5A) was originally designated as an "initiation factor," recent data have shown it to be also involved in apoptosis. However, the actual function of eIF5A in apoptosis is still unknown. In this study, we performed yeast two-hybrid screens to identify eIF5A-interacting proteins to help us understand the mechanisms of eIF5A. Our results demonstrated that eIF5A and syntenin could engage in a specific interaction both in vitro and in vivo and functioned collaboratively to regulate p53 activity. Our findings, for the first time, revealed a new biological activity for eIF5A as the regulator of p53. Overexpression of eIF5A or its EFP domain resulted in up-regulation of p53, and silencing eIF5A by small interfering RNA reduced the p53 protein level. Further analysis by reverse transcription PCR showed eIF5A-activated p53 transcription. The effect of eIF5A on p53 transcriptional activity was further demonstrated by the increasing expressions of p21 and Bax, well known target genes of p53. In contrast, a point mutant of eIF5A, hypusination being abolished, was revealed to be functionally defective in p53 up-regulation. Overexpression of eIF5A led to a p53-dependent apoptosis or sensitized cells to induction of apoptosis by chemotherapeutic agents. However, when eIF5A interacted with its novel partner, syntenin, the eIF5A-induced increase in p53 protein level was significantly inhibited. Therefore, eIF5A seems to be a previously unrecognized regulator of p53 that may define a new pathway for p53-dependent apoptosis, and syntenin might regulate p53 by balancing the regulation of eIF5A signaling to p53 for apoptosis.

Animals↗

PubMed2005

Peroxisome proliferator-activated receptor gamma ligands stimulate endothelial nitric oxide production through distinct peroxisome proliferator-activated receptor gamma-dependent mechanisms.

OBJECTIVE: We recently reported that the peroxisome proliferator-activated receptor gamma (PPARgamma) ligands 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) and ciglitazone increased cultured endothelial cell nitric oxide (NO) release without increasing the expression of endothelial nitric oxide synthase (eNOS). The current study was designed to characterize further the molecular mechanisms underlying PPARgamma-ligand-stimulated increases in endothelial cell NO production. METHODS AND RESULTS: Treating human umbilical vein endothelial cells (HUVEC) with PPARgamma ligands (10 micromol/L 15d-PGJ2, ciglitazone, or rosiglitazone) for 24 hours increased NOS activity and NO release. In selected studies, HUVEC were treated with PPARgamma ligands and with the PPARgamma antagonist GW9662 (2 micromol/L), which fully inhibited stimulation of a luciferase reporter gene, or with small interfering RNA to PPARgamma, which reduced HUVEC PPARgamma expression. Treatment with either small interfering RNA to PPARgamma or GW9662 inhibited 15d-PGJ2-, ciglitazone-, and rosiglitazone-induced increases in endothelial cell NO release. Rosiglitazone and 15d-PGJ2, but not ciglitazone, increased heat shock protein 90-eNOS interaction and eNOS ser1177 phosphorylation. The heat shock protein 90 inhibitor geldanamycin attenuated 15d-PGJ2- and rosiglitazone-stimulated NOS activity and NO production. CONCLUSIONS: These findings further clarify mechanisms involved in PPARgamma-stimulated endothelial cell NO release and emphasize that individual ligands exert their effects through distinct PPARgamma-dependent mechanisms.

Anilides↗

PubMed2006

Clathrin heavy chain is required for TNF-induced inflammatory signaling.

BACKGROUND: Tumor necrosis factor receptor I recruits tumor necrosis factor receptor-associated death domain (TRADD) and multiple kinases that ultimately phosphorylate inhibitor kappa B (IKB alpha). Degradation of phospho-IKB alpha (p-IKB alpha) frees nuclear factor kappa B (NFKB) to be active and phosphorylated. Many receptors require clathrin-mediated endocytosis to provide the scaffolds necessary for signaling. Therefore, we investigated the role of clathrin heavy chain (CHC) in tumor necrosis factor alpha (TNF-alpha)-induced IKB alpha phosphorylation and NFKB activation. We hypothesized that CHC was required for TNF-alpha-induced inflammatory signaling. METHODS: We treated human pulmonary epithelial cells with small interfering RNA to knock down intracellular CHC (CHCsil). TRADD and scrambled (noncoding) small interfering RNA sequences were used as positive and negative controls, respectively. Treatment groups were exposed to 10 ng/mL of TNF-alpha. Total I kappaB alpha, p-I kappaB alpha, and phosphorylated P65 (a subunit of NFKB) were determined by immunoblot staining. Densitometry was normalized to controls for the analysis of the stains. TNF-alpha-induced release of monocyte chemoattractant protein 1 (MCP-1) was determined by enzyme-linked immunosorbent assay. Statistical analyses were determined by analysis of variance or paired t test as appropriate. RESULTS: TNF-alpha-induced I kappaB alpha phosphorylation and degradation at 5 and 30 minutes, respectively, and induced P65 phosphorylation. CHCsil diminished p-I kappaB alpha by 91% (P < .03); however, I kappaB alpha degradation was not affected. CHC knockdown caused a 66% decrease in P65 phosphorylation after 3 minutes of TNF-alpha. CHCsil decreased TNF-alpha-induced MCP-1 by 46% (P < .05), compared with control. CONCLUSIONS: CHCsil significantly impairs phosphorylation of both I kappaB alpha and P65. CHCsil also significantly decreased MCP-1 production. These data suggest that CHC is required for certain TNF-alpha-induced, inflammatory signaling pathways.

Cell Culture Techniques↗

PubMed2005

Small regulatory RNAs in mammals.

Mammalian cells harbor numerous small non-protein-coding RNAs, including small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs) and small double-stranded RNAs, which regulate gene expression at many levels including chromatin architecture, RNA editing, RNA stability, translation, and quite possibly transcription and splicing. These RNAs are processed by multistep pathways from the introns and exons of longer primary transcripts, including protein-coding transcripts. Most show distinctive temporal- and tissue-specific expression patterns in different tissues, including embryonal stem cells and the brain, and some are imprinted. Small RNAs control a wide range of developmental and physiological pathways in animals, including hematopoietic differentiation, adipocyte differentiation and insulin secretion in mammals, and have been shown to be perturbed in cancer and other diseases. The extent of transcription of non-coding sequences and the abundance of small RNAs suggests the existence of an extensive regulatory network on the basis of RNA signaling which may underpin the development and much of the phenotypic variation in mammals and other complex organisms and which may have different genetic signatures from sequences encoding proteins.

Animals↗

PubMed2005

[Effect of RNAi-mediated gene silencing of C-erbB-2 on proliferation of lung adenocarcinoma cell line calu-3].

BACKGROUND & OBJECTIVE: C-erbB-2 gene is amplified or overexpressed in breast cancer, ovarian cancer, and lung cancer, and is related with enhanced malignancy and metastatic ability, intrinsic chemoresistance, and poor prognosis of tumors. RNA interfering (RNAi), a new genetic technique, can efficiently and specifically suppress gene expression. This study was to investigate the effect of small interfering RNA (siRNA)-mediated gene silencing of C-erbB-2 on proliferation of human lung adenocarcinoma cell line calu-3. METHODS: C-erbB-2 siRNA was transfected into calu-3 cells; cell morphology was observed under light microscope. The mRNA and protein levels of C-erbB-2 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM). The proliferation of calu-3 cells was assessed by MTT assay. Cell cycle and apoptosis were analyzed by FCM. RESULTS: C-erbB-2 siRNA down-regulated the mRNA and protein levels of C-erbB-2 in calu-3 cells; 48 h after transfection of C-erbB-2 siRNA, the protein level of C-erbB-2 was markedly decreased. The positive rate of C-erbB-2 was significantly lower in C-erbB-2 siRNA group than in untransfected group, empty vector group, and nonspecific siRNA group [(25.04+/-1.56)% vs. (98.24+/-2.23)%, (95.67+/-1.98)%, and (94.79+/-0.87)%, P < 0.01]. C-erbB-2 siRNA inhibited proliferation of calu-3 cells: G(0)/G(1) phase proportion of C-erbB-2 siRNA group was significantly higher than that of untransfected group [(56.6+/-3.6)% vs. (45.5+/-3.2)%, P < 0.01]. C-erbB-2 siRNA also enhanced cell apoptosis. CONCLUSION: Specific siRNA targeting C-erbB-2 can effectively inhibit C-erbB-2 expression and proliferation of calu-3 cells.

Adenocarcinoma↗

PubMed2005

Protein kinase C-dependent protein kinase D activation modulates ERK signal pathway and endothelial cell proliferation by vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal conditions and in pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are not well defined. Protein kinase D (PKD), a newly described serine/threonine protein kinase, has been implicated in many signal transduction pathways and in cell proliferation. We hypothesized that PKD would mediate VEGF signaling and function in endothelial cells. Here we found that VEGF rapidly and strongly stimulated PKD phosphorylation and activation in endothelial cells via VEGF receptor 2 (VEGFR2). The pharmacological inhibitors for phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) significantly inhibited VEGF-induced PKD activation, suggesting the involvement of the PLCgamma/PKC pathway. In particular, PKCalpha was critical for VEGF-induced PKD activation since both overexpression of adenovirus PKCalpha dominant negative mutant and reduction of PKCalpha expression by small interfering RNA markedly inhibited VEGF-induced PKD activation. Importantly, we found that small interfering RNA knockdown of PKD and PKCalpha expression significantly attenuated ERK activation and DNA synthesis in endothelial cells by VEGF. Taken together, our results demonstrated for the first time that VEGF activates PKD via the VEGFR2/PLCgamma/PKCalpha pathway and revealed a critical role of PKD in VEGF-induced ERK signaling and endothelial cell proliferation.

Adenoviridae↗

PubMed2003

Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing.

Glucose homeostasis is controlled by insulin in part through the translocation of intracellular glucose transporter 4 to the plasma membrane in muscle and fat cells. Akt/protein kinase B downstream of phosphatidylinositol 3-kinase has been implicated in this insulin-signaling pathway, but results with a variety of reagents including Akt1-/- and Akt2-/- mice have been equivocal. Here we report the application of small interfering RNA-directed gene silencing to deplete both Akt1 and Akt2 in cultured 3T3-L1 adipocytes. Loss of Akt1 alone slightly impaired insulin-mediated hexose transport activity but had no detectable effect on glycogen synthase kinase (GSK)-3 phosphorylation. In contrast, depletion of Akt2 alone by 70% inhibited approximately half of the insulin responsiveness. Combined depletions of Akt1 plus Akt2 in these cells even more markedly attenuated insulin action on glucose transporter 4 movements, hexose transport activity, and GSK-3 phosphorylation. These data demonstrate a primary role of Akt2 in insulin signaling, significant functional redundancy of Akt1 and Akt2 isoforms in this pathway, and an absolute requirement of Akt protein kinases for regulation of glucose transport and GSK-3 in cultured adipocytes.

3T3 Cells↗

PubMed2005

RNA interference in vivo: toward synthetic small inhibitory RNA-based therapeutics.

Small interfering RNA (siRNA) mediated inhibition of gene expression has rapidly become a major tool for in vitro analysis of protein function. In vivo gene silencing by siRNAs will play an important role for target validation and is the first step towards the development of siRNA-based therapeutics. This chapter reviews the early and intriguing successes in using siRNAs for in vivo gene silencing. The impact of chemical modification on siRNA efficacy in vitro and the potential for employing such modifications to alter the pharmacokinetic properties of siRNAs is also summarized. A protocol describing siRNA-based gene silencing in tumor models can serve as guide for the design of individual in vivo RNA interference experiments.

Base Sequence↗

PubMed2005

GIT1 is a scaffold for ERK1/2 activation in focal adhesions.

GIT1 (G protein-coupled receptor kinase-interacting protein 1) has been shown to regulate focal adhesion disassembly. We previously reported that GIT1 associates with MEK1 and acts as a scaffold to enhance ERK1/2 activation. Here, we show that GIT1 co-localizes with ERK1/2 in focal adhesions and regulates cell migration in vascular smooth muscle cells, HEK293 cells, and HeLa cells. Immunofluorescence showed that GIT1 co-localized with phospho-ERK1/2 in focal adhesions after epidermal growth factor stimulation. Because Src is required for both GIT1 tyrosine phosphorylation and focal adhesion disassembly, we studied the effects of Src on GIT1-ERK1/2 interactions. PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) inhibited association of GIT1 with ERK1/2, and their co-localization in focal adhesions was dramatically decreased in SYF-/- cells. GIT1 small interfering RNA significantly inhibited ERK1/2 recruitment to and activation in focal adhesions. GIT1 small interfering RNA and mutated GIT1 lacking the MEK1 binding domain significantly decreased epidermal growth factor-stimulated cell spreading and migration, suggesting that GIT1-mediated events such as ERK1/2 activation are required for spreading and migration. In summary, the present study further supports a key role for GIT1 (a MEK1-binding protein) as a scaffold for signal transduction in focal adhesions.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Generation of variable and fixed length siRNA from a novel siRNA expression vector.

Small interfering RNA (siRNA) expression vectors using a Pol III promoter fall into two categories, vectors with a single Pol III promoter that express small hairpin RNA (shRNA) and vectors with two head-to-head (convergent) Pol III promoters that express siRNA. There are technical difficulties in preparing convergent siRNA vectors from cDNA. Here, we report construction of a novel convergent siRNA expression vector, pTHUB. Two XcmI sites were inserted between opposing Pol III promoters. After linearization with XcmI, pTHUB has a single 3' A overhang at each end that allows direct cloning of partially DNase I digested cDNA fragments (20-30 bp) tailed with ddT. A derivative method for generating 19 bp siRNA in pTHUB is also described. The suppression efficiency of the pTHUB vector is comparable to those of conventional shRNA vectors. We have made a siRNA library from a single cDNA. The same approach can be used to construct whole-genome siRNA libraries from cellular cDNA.

Base Sequence↗

PubMed2005

Quantum dot-conjugated hybridization probes for preliminary screening of siRNA sequences.

In the present study, we describe the design and fabrication of quantum dot-conjugated hybridization probes and their application to the development of a comparatively simple and rapid procedure for the selection of highly effective small-interfering RNA (siRNA) sequences for RNA interference (RNAi) in mammalian cells, for example, siRNAs with high accessibility and affinity to the respective mRNA target. A single-stranded siRNA was conjugated with a quantum dot and used as a hybridization probe. The target mRNA was amplified in the presence of Cy5-labeled nucleotides, and Cy5-mRNA served as a hybridization sample. The formation of siRNA/mRNA duplexes during a comparatively short hybridization time (1 h) was used as a criterion for the selection of highly effective, target-specific siRNA sequences. The accessibility and affinity of the siRNA sequence for the target mRNA site were determined by fluorescence resonance energy transfer (FRET) between a quantum dot (donor) and a fluorescent dye molecule (Cy5, acceptor) localized at an appropriate distance from each other when hybridization occurred. The FRET signal was observed only when there was high accessibility between an antisense siRNA and a sense mRNA and did not appear in the case of mismatch siRNAs. Moreover, the amplitude of the FRET signal significantly correlated with the specific effect of siRNA on the expression of the target mRNA and protein, determined in native cells by RT-PCR and immunoblot analysis, respectively.

Base Sequence↗

PubMed2005

[Construction of eukaryotic expression vector of siRNA specific to bcr/abl fusion gene].

OBJECTIVE: To construct eukaryotic expression vector of siRNA specific to bcr/abl and to initially investigate the effect of recombinant plasmid on bcr/abl and P210 protein expression in K562 cells. METHODS: siRNA (small interfering RNA) was designed according to the Tuschl's principle of RNAi-based medicine, and was converted into cDNA coding expression of shRNA (small hairpin RNAs)of siRNA for bcr/abl fusion gene. The cDNA was synthesized and inserted into plasmid pTER. The pTER117 and pTER363 of recombinant plasmid being eukaryotic expression vector was controlled by the H1 promoter of RNA polymerase III, identified by the restriction map and the sequence analysis, and transfected into K562 cells by Lipofectamine. Expression of bcr/abl mRNA was assayed by RT-PCR; expression of P210 protein was detected by immunohistochemistry. RESULTS: The pTER117 and pTER363 of recombinant plasmid identified by the restriction map and the sequence analysis completely coincided with the designs. 24 hours after transfection in K562 cells, the recombinant plasmid could down regulate the expression of the bcr/abl mRNA and bcr/abl protein(P210) in K562 cells. CONCLUSION: The siRNA eukaryotic expression vector against bcr/abl mRNA has been successfully conctructed, and it effectively inhibits the expression of bcr/abl in K562 cells.

Base Sequence↗

PubMed2004

A new vector, based on the PolII promoter of the U1 snRNA gene, for the expression of siRNAs in mammalian cells.

Several vectors for the induction of RNA interference in mammalian cells have been described,based mainly on polIII-dependent promoters. They transcribe short hairpin RNAs (shRNA) that,after being processed into short interfering RNAs (siRNAs), mediate the degradation of the target mRNA. Here, we describe the construction of a new siRNA-expressing vector (psiUx) based on the strong and ubiquitous polII-dependent promoter of the human U1 small nuclear RNA (snRNA)gene. In psiUx, the only constraint for the shRNA sequence is a purine at position +1, since specific 3'-end formation is achieved by a box element located downstream of the transcribed region. Several constructs were designed against the lamin A/C target. Depending on the structure of the shRNA transcribed, a preferential or exclusive accumulation of the antisense strand is obtained, thus avoiding possible nonspecific targeting by the sense strand. In all cases tested, very effective siRNAs were produced, thus providing a proof-of-principle that a snRNA-type polII promoter can be used for the expression of siRNAs. We show that psiUx ensures high levels of expression and efficient knock down of the target gene also in stable cell lines.

Genetic Vectors↗