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At least 415 records · Page 23Linked to original sources
PubMed2006

RNAi-mediated downregulation of urokinase plasminogen activator and its receptor in human meningioma cells inhibits tumor invasion and growth.

In recent years, RNA interference (RNAi) has emerged as an effective method to target specific genes for silencing. Several groups are actively exploring the use of small interfering RNA (siRNA) for therapeutic applications to treat cancer. Our previous studies have demonstrated the inhibition of various proteases, including serine proteases, cysteine proteases and matrix metalloproteases, via RNA interference (RNAi) in gliomas. Similar to gliomas, malignant meningiomas also exhibit elevated protease levels in comparison to normal brain and benign meningiomas. Here, we used siRNA to simultaneously target urokinase plasminogen activator (uPA) and its receptor, uPAR. A human CMV promoter-driven mammalian expression vector (pU2) was used to produce hairpin double-stranded RNA (hp RNA) to target uPA and uPAR. As determined by Western blotting and fibrin zymography, pU2 effectively inhibited uPAR protein levels and uPA enzymatic activity in meningioma cells (IOMM-Lee). In vitro studies (Matrigel invasion and spheroid migration) revealed reduced meningioma cell invasion and migration. Intratumoral injections of the plasmid vector expressing siRNA for uPA and uPAR resulted in regression of pre-established, subcutaneous tumors in mice. In addition, in vivo studies of mice injected with pU2-transfected meningioma cells revealed inhibition of intracranial tumor formation. These findings suggest that siRNA can be used as a potent and specific therapeutic tool for the treatment of malignant meningiomas in humans.

Animals↗
PubMed2006

Effects of RNAi-mediated Smad4 silencing on growth and apoptosis of human rhabdomyosarcoma cells.

Transforming growth factor-beta (TGF-beta) signals through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, TGF-beta activates intracellular Smad proteins and regulates proliferation and apoptosis in various cell types. To demonstrate the effects of TGF-beta/Smad signal on growth and apoptosis of human embryonal rhabdomyosarcoma (RMS) cells, a strategy of RNAi-mediated 'gene silencing' of Smad4 was used to interrupt endogenous TGF-beta/Smad signaling in an RMS cell line, RD, and the regulation of exogenous TGF-beta1 to growth and apoptosis of the cells was also determined. Physiologically, TGF-beta/Smad signaling was essential for the normal growth of RD. The interruption of endogenous TGF-beta/Smad signaling by RNAi significantly suppressed the growth of RD cells and dramatically induced apoptosis of RD cells. Exogenous TGF-beta1 also inhibited the growth of RD cells, but had no effect on apoptosis. It also partially counteracted the growth inhibition and apoptosis induced by Smad4 silencing in RD cells. These findings provide a new insight into how TGF-beta/Smad signaling regulates the growth and apoptosis of cancer cells. Moreover, as a powerful tool, shRNA interference suppresses endogenous Smad4 gene expression and subsequently modulates cell growth and apoptosis, which may provide a novel basis for the development of rational intervention strategies in RMS therapy.

Apoptosis↗
PubMed2006

[Knocking down rat Mecp2 expression by RNAi].

OBJECTIVE: To find the valid siRNA (small interference RNA) sequence to knock down rat Mecp2 expression for the analysis of Mecp2 function by RNA interference (RNAi). METHODS: A plasmid (pMecp2-RFP) expressing rat Mecp2 and marker gene red fluorescent protein (RFP) as a fusion gene was constracted. We first selected a candidate valid sequence by cotransfecting the pMecp2-RFP with 4 candidate siRNAs targeting rat Mecp2 to HEK293T cells respectively, and then identified the RNAi efficiency of the candidate valid siRNA by detecting its effect on suppressing the expression of endogenous rat Mecp2 in PC12 cells. RESULTS: siRNA sequence (5'-GCUGUGAAGGAAUCUUCUA-3') targeting rat Mecp2 mRNA 918-936nt was the valid sequence to knock down rat Mecp2 expression by RNA interference. As the target sequence was located in exon 4 of rat Mecp2 mRNA, we assumed that it could suppress the expression of Mecp2alpha and Mecp2beta at the same time. And as the target sequence was located in the coding region of rat Mecp2, we assumed that it could suppress the expression of both 1.9 kb and 10 kb of rat Mecp2 transcript. CONCLUSION: This study has laid the foundation for constructing rat Mecp2 knocked-down neuronal cell model to study the gene function of Mecp2 in brain development.

Animals↗
PubMed2006

RNA interference (RNAI) as a tool to engineer high nutritional value in chicory (Chicorium intybus).

The major component of chicory (Chicorium intybus) root is inulin, which is a polymer of fructose. Inulin production from chicory is hampered by the enzyme fructan 1-exohydrolase (1-FEH) that degrades inulin and limits its yield. Increased FEH activity results in massive breakdown of fructan and production of Fructose and inulo-n-oses. The latter phenomena are to be avoided for industrial fructan production. RNA silencing, which is termed post-transcriptional gene silencing (PTGS) in plants, is an RNA degradation process through sequence specific nucleotide interactions induced by double-stranded RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and co-suppression, in terms of its efficiency and stability. We are generating a transgenic chicory plants with suppressed FEH (exohydrolas) genes using RNAi resulting in supressed inulin degradation. A small but important part of the construct is a sequence unique for the target gene (exons) or genes,which were cloned. The hairpin constructs were made and chicory was transformed by Agrobacterium tumifaciense, strain (C58C1). The transgenics should be select and check by means of molecular techniques.

Carbohydrate Metabolism↗
PubMed1987

[4,5-S-RNAI in virus-specific polyribosomes from ascitic Krebs 2 carcinoma cells infected with encephalomyocarditis virus].

Polyribosomes of Krebs 2 ascite carcinoma cells non-infected and infected with encephalomyocarditis (EMC) virus contain a heterogeneous population of low molecular weight small RNAs. Analysis of the RNAs by polyacrylamide gel electrophoresis did not reveal any qualitative differences in the small RNA sets within the composition of polyribosomes from virus-infected and non-infected cells. However, the content of one of the small RNAs was markedly elevated in polyribosomes from virus-infected cells. As can be followed from partial determination of its primary structure, this small mRNA is identical to 4,5S-RNAI previously detected in the nuclei of Novikov hepatoma cells of the rat. The data obtained suggest that 4,5S-RNAI can be involved in the regulation of protein synthesis in virus-infected cells.

Animals↗
PubMed2006

Engineering eukaryotic signal transduction with RNAi: enhancing Drosophila S2 cell growth and recombinant protein synthesis via silencing of TSC1.

RNAi has been useful in the study of biochemical pathways, but has not been widely used as a tool in metabolic engineering. The work described here makes use of double-stranded RNA (dsRNA) for the post-transcriptional gene silencing of TSC1 in Drosophila S2 cells. TSC1 downregulates the insulin-mediated signal transduction pathway, and serves as a metabolic control to guard against cellular overproliferation and tumorogenesis in both flies and mammals. By silencing TSC1 with in vitro-synthesized dsRNA, we have created a tunable and specific metabolic "throttle" that, like insulin, apparently increases the specific growth rate of S2 cells in a dose-dependent manner. This "throttle," augments the benefits of insulin addition while apparently avoiding deleterious and pleiotropic effects which can lead to lysis. During the period wherein dsRNA was active, cell growth rate was increased by 11% by the addition of 15 microg/mL dsTSC1 and by over 20% by the addition of 30 microg/mL dsTSC1. Additionally, synthesis of recombinant green fluorescent protein (GFP) was increased nearly 50% in a stable S2 cell line inducibly expressing GFP. Accordingly, we have "tuned" a normally tumorogenic pathway in animals into an advantage for both growth and recombinant product synthesis in cell culture. Potential applications for improving eukaryotic cell culture are anticipated.

Animals↗
PubMedDate not supplied

Blocking BE301622 gene expression by RNAi initiates differentiation of neural stem cells in rat.

Neural stem cells (NSCs) are capable of differentiating into neurons, astrocytes and oligodendrocytes. However, the molecular mechanisms regulating NSCs differentiation are not well understood. Our previous research by microarray analysis certified that a lot of genes are differentially expressed in the course of NSC differentiation. In this study we report the function of one of these genes, BE301622, by RNAi techniques. To silence the BE301622 gene, a long, double-stranded RNA (dsRNA) was synthesized by using a kit (Ambion T7 MegaScript) and transformed into NSCs. Expression of mRNA was tested through RT-PCR. The result showed the expression of BE301622 gene was specificially suppressed. This finding effectively validated that BE301622 is involved in the differentiation of NSCs.

Animals↗
PubMed2007

Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes.

Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.

Animals↗
PubMed2006

Effective inhibition of cell growth and invasion of melanoma by combined suppression of BRAF (V599E) and Skp2 with lentiviral RNAi.

p27Kip1 that regulates the G1/S transition of cell cycle and inhibits Rho A signaling is frequently lost in several cancers leading to the deregulation of cell growth and cell motility. Mitogen-activated protein kinases (MAPK) regulate the export of p27Kip1 from nucleus to cytoplasm, followed by the degradation with proteases. Skp-2, a subunit of an SCF ubiquitin-protein ligase complex responsible for the ubiquitination of p27Kip1, is upregulated frequently in several cancers, leading to the decrease of p27Kip1. We applied human immunodeficiency virus (HIV) lentivirus-mediated RNA interference (RNAi) to melanoma cells with the BRAF mutation (V599E) and overexpressed Skp-2 and found that the simultaneous suppression of these activated oncogenes resulted in the effective inhibition of in vitro cell growth and invasive ability of melanoma cells accompanied by the additional increase of p27Kip1. Our results suggest that gene therapy against melanoma with the enhanced MAPK and ubiquitin-proteasomal pathways could be a specific and effective therapeutic strategy for cancers.

Activin Receptors, Type I↗
PubMed2006

RNAi-mediated silencing of insulin receptor substrate 1 (IRS-1) enhances tamoxifen-induced cell death in MCF-7 breast cancer cells.

Insulin receptor substrate 1 (IRS-1) is a major downstream signaling protein for insulin and insulin-like growth factor I (IGF-I) receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In breast cancer, IRS-1 overexpression has been associated with tumor development, hormone-independence and antiestrogen-resistance. In part, these effects are related to potentiation of IRS-1/PI-3K/Akt signaling. In estrogen sensitive breast cancer cell lines, tamoxifen treatment reduces IRS-1 expression and function; consequently, inhibiting IRS-1/PI-3K signaling. We tested whether anti-IRS1 siRNA could inhibit growth and survival of estrogen-sensitive MCF-7 breast cancer cells, when used alone or in combination with TAM. Our results indicated: (a) out of four tested anti-IRS1 siRNAs, two siRNAs reduced IRS-1 protein by approximately three-fold in both growing and IGF-I-stimulated cells without affecting a closely related protein, IRS-2; (b) these effects paralleled IRS1 mRNA downregulation by approximately three-fold, measured by quantitative real time-polymerase chain reaction; (c) action of anti-IRS1 siRNAs induced the apoptotic response, observed by altered mitochondrial membrane potential coupled with downregulation of NF-kappaB target Bcl-xL and reduced cell viability; (d) anti-IRS1 siRNA treatment enhanced the cytotoxic effects of TAM by approximately 20%. In summary, anti-IRS1 RNAi strategy could become a potent tool to induce breast cancer cell death, especially if combined with standard TAM therapy.

Breast Neoplasms↗
PubMed2006

Antiviral applications of RNAi.

RNA interference is a natural mechanism by which small interfering (si)RNA operates to specifically and potently down-regulate the expression of a target gene. This down-regulation has been thought to predominantly function at the level of the messenger (m)RNA, post-transcriptional gene silencing (PTGS). Recently, the discovery that siRNAs can function to suppress a gene's expression at the level of transcription, i.e., transcriptional gene silencing (TGS), has created a major paradigm shift in mammalian RNAi. These recent findings significantly broaden the role RNA, specifically siRNAs and potentially microRNAs, plays in the regulation of gene expression as well as the breadth of potential siRNA target sites. Indeed, the specificity and simplicity of design makes the use of siRNAs to target and suppress virtually any gene or gene promoter of interest a realized technology. Furthermore, since siRNAs are a small nucleic acid reagent, they are unlikely to elicit an immune response, making them a theoretically good future therapeutic. This review will focus on the development, delivery, and potential therapeutic use of antiviral siRNAs in treating viral infections as well as emerging viral threats.

Animals↗
PubMed2006

RNAi-mediated knockdown of aldehyde dehydrogenase class-1A1 and class-3A1 is specific and reveals that each contributes equally to the resistance against 4-hydroperoxycyclophosphamide.

PURPOSE: Aldehyde dehydrogenases class-1A1 (ALDH1A1) and class-3A1 (ALDH3A1) have been associated with resistance to cyclophosphamide (CP) and its derivatives. We have previously reported the downregulation of these enzymes by all-trans retinoic acid (ATRA). METHODS: In this study, we used siRNA duplexes as well as retrovirally expressed siRNA to knockdown one or both enzymes together in A549 lung cancer cell line in order to investigate the role of each one in mediating the resistance and the effect of the addition of ATRA. RESULTS: The results show that significant and specific knockdown of each enzyme can be achieved and that each one contributes similarly to cell resistance to 4-hydroperoxycyclophosphamide (4-HC), an active derivative of CP. Added effects were seen when both enzymes were inhibited. The addition of ATRA also exhibited additional inhibitory effects on ALDH activity and increased 4-HC toxicity when added to single siRNA aimed at one of the enzymes. On the other hand, ATRA had minimal and insignificant additional inhibitory effects on ALDH enzyme activity when added to a combination of siRNAs against both enzymes, but still increased 4-HC toxicity beyond that seen with RNAi-mediated inhibition of both enzymes together. CONCLUSIONS: We conclude that both enzymes, ALDH1A1 and ALDH3A1 will need to be blocked in order to achieve the highest sensitivity to 4-HC. Furthermore, ATRA increases 4-HC toxicity even when added to a combination of siRNAs against both enzymes, thus suggesting additional mechanisms by which ATRA can increase drug toxicity.

Actins↗
PubMed2003

Characterization of two rice DNA methyltransferase genes and RNAi-mediated reactivation of a silenced transgene in rice callus.

Two genomic clones ( OsMET1-1, AF 462029 and OsMET1-2, TPA BK001405), each encoding a cytosine-5 DNA methyltransferase (MTase), were isolated from rice ( Oryza sativa L.) BAC libraries. OsMET1-1 has an open reading frame of 4,566 nucleotides with 12 exons and 11 introns while OsMET1-2 has an open reading frame of 4,491 nucleotides with 11 exons and 10 introns. Although OsMET1-1 and OsMET1-2 have high sequence similarity overall, they share only 24% identity in exon 1, and intron 3 of OsMET1-1 is absent from OsMET1-2. As for other eukaryotic DNA MTases of the Dnmt1/MET l class, the derived amino acid sequences of OsMET1-1 and OsMET1-2 suggest that they are comprised of two-thirds regulatory domain and one-third catalytic domain. Most functional domains identified for other MTases were present in the rice MET1 sequences. Amino acid sequence comparison indicated high similarity (56-75% identity) of rice MET1 proteins to other plant MET1 sequences but limited similarity (approx. 24% identity) to animal Dnmt1 proteins. Genomic blot and database analysis indicated the presence of a single copy of OsMET1-1 (on chromosome 3) and single copy of OsMET1-2 (on chromosome 7). Ribonuclease protection assays revealed expression of both OsMET1-1 and OsMET1-2 in highly dividing cells, but the steady-state level of OsMET1-2 was 7- to 12-fold higher than that for OsMET1-1 in callus, root and inflorescence. The functional involvement of the rice DNA MTases in gene silencing was investigated using an RNAi strategy. Inverted repeat constructs of either the N- or C-terminal regions of OsMET1-1 were supertransformed into calli derived from a rice line bearing a silenced 35S-uidA-nos transgene. Restoration of uidA expression in the bombarded calli was consistent with the inactivation of maintenance methylation and with previous evidence for the involvement of methylation in silencing of this line.

Base Sequence↗
PubMed2006

RNAi-mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content.

Inositol plays a role in membrane trafficking and signaling in addition to regulating cellular metabolism and controlling growth. In plants, the myo-inositol-1-phosphate is synthesized from glucose 6-phosphate in a reaction catalyzed by the enzyme myo-inositol-1-phosphate synthase (EC 5.5.1.4). Inositol can be converted into phytic acid (phytate), the most abundant form of phosphate in seeds. The path to phytate has been suggested to proceed via the sequential phosphorylation of inositol phosphates, and/or in part via phosphatidylinositol phosphate. Soybean [Glycine max (L.) Merrill] lines were produced using interfering RNA (RNAi) construct in order to silence the myo-inositol-1-phosphate (GmMIPS1) gene. We have observed an absence of seed development in lines in which the presence of GmMIPS1 transcripts was not detected. In addition, a drastic reduction of phytate (InsP6) content was achieved in transgenic lines (up to 94.5%). Our results demonstrated an important correlation between GmMIPS1 gene expression and seed development.

Cotyledon↗
PubMed2007

RNAi-induced phenotypes suggest a novel role for a chemosensory protein CSP5 in the development of embryonic integument in the honeybee (Apis mellifera).

Small chemosensory proteins (CSPs) belong to a conserved, but poorly understood, protein family found in insects and other arthropods. They exhibit both broad and restricted expression patterns during development. In this paper, we used a combination of genome annotation, transcriptional profiling and RNA interference to unravel the functional significance of a honeybee gene (csp5) belonging to the CSP family. We show that csp5 expression resembles the maternal-zygotic pattern that is characterized by the initiation of transcription in the ovary and the replacement of maternal mRNA with embryonic mRNA. Blocking the embryonic expression of csp5 with double-stranded RNA causes abnormalities in all body parts where csp5 is highly expressed. The treated embryos show a "diffuse", often grotesque morphology, and the head skeleton appears to be severely affected. They are 'unable-to-hatch' and cannot progress to the larval stages. Our findings reveal a novel, essential role for this gene family and suggest that csp5 (unable-to-hatch) is an ectodermal gene involved in embryonic integument formation. Our study confirms the utility of an RNAi approach to functional characterization of novel developmental genes uncovered by the honeybee genome project and provides a starting point for further studies on embryonic integument formation in this insect.

Amino Acid Sequence↗
PubMed2004

Use of RNAi technology to confer enhanced resistance to BmNPV on transgenic silkworms.

dsRNA is a powerful tool for gene-specific silencing in plants and animals. In this study, we examined the use of gene silencing in generating transgenic silkworms resistant to the Bombyx mori nucleopolyhedrovirus (BmNPV). Using a transposon piggyBac system, we first generated BmN cells (rBmN-lef1), which carried artificial genes designed for expressing dsRNAs with sequences of the essential viral gene lef-1. NPV DNA microarray analysis revealed that the accumulation of lef-1 mRNA was successfully inhibited in rBmN-lef1 infected with BmNPV. The virus titer in the culture medium of rBmN-lef1 at 48 hr post-infection (h.p.i.) was 50% of that of the control cells. Moderate BmNPV-resistance caused by transgenesis of the artificial dsRNA-expressing gene was confirmed in the transgenic silkworms. Virus production was reduced in transgenic silkworms relative to controls up to 96 hrs after viral inoculation. Although complete protection was not achieved and the transgenic larvae ultimately died, this is the first report to show the use of RNAi in confering enhanced viral resistance on transgenic animals.

Animals↗
PubMed2006

A principal role for AtXTH18 in Arabidopsis thaliana root growth: a functional analysis using RNAi plants.

Rearrangement of cellulose microfibrils within cell-wall matrices is considered one of the most critical steps in the regulation of both the orientation and extent of cell expansion in plants. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a family of enzymes that mediate the construction and restructuring of load-bearing cross links among cellulose microfibrils. The Arabidopsis thaliana XTH genes AtXTH17, 18, 19, and 20 are phylogenetically closely related to one another and are preferentially expressed in the roots. However, they exhibit different expression profiles within the root and respond to hormonal signals differently. To investigate their functions in root growth, we examined phenotypes of loss-of-function mutants for these genes using T-DNA insertion lines and RNAi plants. These functional analyses disclosed a principal role for the AtXTH18 gene in primary root elongation. Of the four XTH genes, AtXTH18 exhibits the highest level of mRNA expression. We also determined auxin-signaling pathways for these genes using a mutant with a defect in the AXR2/IAA7 gene and found that the expression of AtXTH19 in the elongation/maturation region of the root is under the control of the AXR2/IAA7 signaling pathway.

Arabidopsis↗
PubMed2006

Characterization of T-DNA insertion mutants and RNAi silenced plants of Arabidopsis thaliana UV-damaged DNA binding protein 2 (AtUV-DDB2).

The human UV-damaged DNA binding protein (UV-DDB), a heterodimeric protein composed of 127 kDa (UV-DDB1) and 48 kDa (UV-DDB2) subunits, has been shown to be involved in DNA repair. To elucidate the in vivo function of plant UV-DDB2, we have analyzed T-DNA insertion mutants of the Arabidopsis thaliana UV-DDB2 subunit (atuv-ddb2 mutants) and AtUV-DDB2 RNAi silenced plants (atuv-ddb2 silenced plants). atuv-ddb2 mutants and atuv-ddb2 silenced plants were both viable, suggesting that AtUV-DDB2 is not essential for survival. Interestingly, both plant types showed a dwarf phenotype, implying impaired growth of the meristem. To the best of our knowledge, this is the first occasion that a dwarf phenotype has been found to be associated with a UV-DDB2 mutation in either plants or animals. The mutants also demonstrated increased sensitivity to UV irradiation, methyl methanesulfonate and hydrogen peroxide treatment, indicating that AtUV-DDB2 is also involved in DNA repair. Our results lead us to suggest that not only does AtUV-DDB2 function in DNA repair, it also has a direct or indirect influence on cell proliferation in the plant meristem.

Arabidopsis↗