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At least 559 records · Page 31Linked to original sources
PubMed2006

Construction of shRNA targeted to the rat angiotensin II type 1 receptors and its RNAi in cytoplasma.

The expression vector of shRNA targeted to the rat angiotensin II receptor gene was constructed and the efficacy of siRNAs to modulate the expression of target gene in the in vitro cultured mammalian cells was investigated for antihypertensive therapy in spontaneous hypertensive rat (SHR) at post-transcriptional level. The sense and antisense RNA oligonucleotides strands targeting angiotensin II receptor mRNA were synthesized individually according to the sequence of the rat angiotensin II receptor. For preparation of duplexes, sense- and antisense-stranded oligonucleotides were mixed and annealed, and the annealed duplexes were cloned into the pGenesil-1 vector. . The rat glioma cells were transfected with constructed pGenesil-1-shRNA plasmid and scrambled plasmid. The cultured cells were collected at different phases. RT-PCR and Western blot were performed. The AT1 mRNA and protein levels behaved ultimately same. Compared to control after 48 h, AT1 mRNA levels were decreased to 35.5% +/- 3.0%, and the levels reached their lowest point after 72 h (20.7% +/- 4% of control). At 24 and 48 h, AT1 protein was reduced to 46.9% +/- 4. 2% and 36.98% +/- 3.7% respectively compared to control and a maximum reduction was observed after 72 h of incubation (28.1% +/- 4% compared to controls). Plasmid-based shRNA expression systems targeted against the rat angiotensin II receptor gene were generated successfully. The shRNAs with a 22-nt stem and a short loop were cleaved into small interfering dsRNA (siRNA) by the Dicer. The in vitro transcribed siRNA enables the effective silencing of gene expression to the target mRNA and leads to effective inhibition of translation of proteins and will be lay the foundation of application of gene silencing technology to hypertensive rats.

Angiotensin II↗
PubMed2006

RNAi-based tuning of cell cycling in Drosophila S2 cells--effects on recombinant protein yield.

We have demonstrated the RNA interference-based interruption of cellular controllers to increase recombinant protein yield in Drosophila Schneider 2 (S2) cell culture. Double-stranded RNA (dsRNA) was enzymatically synthesized in vitro and transfected into stable cell lines expressing green fluorescent protein (GFP) under an inducible promoter. Components of cell cycling (CycE and ago) were silenced with dsRNA homologous to a 700-nucleotide section of their respective mRNA transcripts. Silencing ago and CycE resulted in increases in product yield of up to 1.8-fold and 4-fold, respectively, relative to a control transfected with nuclease-free water. It is surprising to note that nearly complete silencing of CycE resulted in no significant change in GFP fluorescence after 24 h, and a decrease in fluorescence after 72 h. By partially silencing CycE, however, we were able to retain 80% of the cells in G1 (48-h sample) and increase GFP synthesis by fourfold. Implications for protein synthesis processing are discussed.

Animals↗
PubMed2006

RNAi, microRNAs, and human disease.

MicroRNAs (miRNAs) are short, noncoding RNAs that posttranscriptionally regulate gene expression. Over 300 miRNA genes have been identified in the human genome. We have undertaken the study of miRNA function in mammals. Using a custom microarray platform, we investigated miRNA expression patterns in mammalian development and in cancer. We found that many miRNAs are downregulated in cancer. On the other hand, several miRNA genes are overexpressed in tumor cell lines and primary tumors. Seven of these cancer-associated miRNAs are clustered in a single primary transcript termed chr13orf 25 or OncomiR-1. This cluster is located in a region amplified in lymphoma and several solid malignancies. Ectopic expression of these miRNAs in a mouse model of lymphoma accelerated disease progression. In addition, the lymphomas had reduced apoptosis and were more disseminated into secondary regions. This work establishes noncoding RNAs, and specifically miRNAs, as oncogenes in human cancers.

Humans↗
PubMed2006

Transgenes consisting of a dsRNA of an RNAi suppressor plus the 3' UTR provide resistance to Citrus tristeza virus sequences in Nicotiana benthamiana but not in citrus.

In an attempt to utilize post-transcriptional gene silencing (PTGS) as a means to impart resistance against Citrus tristeza virus (CTV) into citrus plants, the p23 + 3'UTR sequence (p23U) of the VT strain of CTV was engineered to fold into a double-stranded (ds) RNA structure. The resulting construct (p23UI) was introduced into Nicotiana benthamiana and Alemow (Citrus macrophylla) plants by Agrobacterium-mediated transformation. Transgenic p23UI- N. benthamiana were resistant to infection with a viral vector made of Grapevine virus A (GVA) + p23U (GVA-p23U), as indicated by the absence of the chimeric virus from inoculated plants. Inoculation of transgenic p23UI Alemow plants with CTV resulted in delayed appearance of symptoms in 9 out of the 70 transgenic plants. However, none of the plants showed durable resistance, as indicated by the obtaining of similar Northern hybridization signals from both transgenic and non-transgenic citrus plants. The possible causes for the failure of transgenic citrus plants to confer durable resistance to CTV are discussed.

3' Untranslated Regions↗
PubMed2006

Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53.

Drug resistance to 5-fluorouracil (5-FU) is still a major limitation to its clinical use. In addition, the clinical value of p53 as a predictive marker for 5-FU-based chemotherapy remains a matter of debate. Here, we used HCT116 human colorectal cancer cells expressing wild-type p53 and investigated whether inhibition of Fas expression by interference RNA modulates 5-FU-induced apoptosis. Cells were treated with 5-FU (1, 4 or 8 microM) for 8-48 h. Cell viability was evaluated by trypan blue dye exclusion. Apoptosis was assessed by changes in nuclear morphology and caspase activity. The interference RNA technology was used to silence Fas expression. Caspase activation, p53, Fas, cytochrome c, and Bcl-2 family protein expression was evaluated by immunoblotting. 5-FU was cytotoxic in HCT116 cells (p<0.001). Nuclear fragmentation and caspase-3, -8 and -9 activities were also markedly increased in HCT116 cells after 5-FU (p<0.001). In addition, wild-type p53 and Fas expression were 25- and 4-fold increased (p<0.05). Notably, when interference RNA was used to inhibit Fas, 5-FU-mediated nuclear fragmentation and caspase activity were markedly reduced in HCT116 cells. Finally, western blot analysis of mitochondrial extracts from HCT116 cells exposed to 5-FU showed a 6-fold increase in Bax, together with a 3-fold decrease in cytochrome c (p<0.001). In conclusion, 5-FU exerts its cytotoxic effects, in part, through a p53/Fas-dependent apoptotic pathway that involves Bax translocation and mitochondrial permeabilization.

Antimetabolites, Antineoplastic↗
PubMed2004

Gene silencing using a heat-inducible RNAi system in Arabidopsis.

Controlling gene expression during plant development is an efficient tool to explore gene function. In this paper, we describe a gene expression system driven by a heat-shock gene promoter (HSP18.2), to trigger the expression of an intron-containing inverted-repeat. RNA interference became a powerful way for gene functional analysis by reverse genetic approaches. However, constitutive gene silencing cannot be used with genes involved in fundamental processes such as embryo viability. Inducible promoters provide an alternative approach for temporal and spatial gene expression control and we described here a new system, complementary to those using chemical gene inducers. To evaluate the efficiency of this system, RNA corresponding to the phytoene desaturase gene of Arabidopsis thaliana was used as a reporter gene in transgenic plants and a comparative study was performed using either the CaMV35S constitutive promoter or the HSP18.2 inducible promoter.

Arabidopsis↗
PubMed2005

S100A4 inhibition by RNAi up-regulates osteoblast related genes in periodontal ligament cells.

Periodontal ligament (PDL) is a thin fibrous connective tissue located between alveolar bone and cementum that remains unmineralized physiologically. It is thus thought that PDL cells possess mechanisms to inhibit mineralization. It has been demonstrated that S100A4, a member of the S100 calcium-binding protein family, is synthesized and secreted by PDL cells, and that it may act as an inhibitor of mineralization. However, the mechanisms of action of S100A4 in mineralization have not been thoroughly clarified. In the present study we investigated the effects of S100A4 inhibition by a short interfering RNA (siRNA) on the expression of osteoblast related genes by human PDL cells. Inhibition of S100A4 by siRNA resulted in increased expression of osteoblastic markers such as osteopontin and osteocalcin, and the osteoblast-specific transcription factors, Runx2/Cbfa1 and Osterix. These results indicate that S100A4 suppresses the expression of osteoblastic genes in PDL cells and may thus inhibit mineralization in the PDL.

Cell Differentiation↗
PubMed2005

Depletion of CPAP by RNAi disrupts centrosome integrity and induces multipolar spindles.

We previously identified a novel microtubule-destabilizing motif in CPAP that can disassemble microtubules. To examine further the CPAP function in human cells, we used siRNA to knockdown its expression. Our results showed that CPAP depletion arrested cells in mitosis and induced apoptosis. Interestingly, more than 40% of these mitotic cells had multiple spindle poles. Furthermore, inhibition of the kinesin Eg5 in CPAP-depleted cells resulted in monopolar spindles, indicating that Eg5 function is required for multipolar spindle formation in the absence of CPAP. Together, our results reveal a structural role for CPAP to maintain centrosome integrity and normal spindle morphology during cell division.

Apoptosis↗
PubMed2006

RNAi-mediated inhibition of the glucosylceramide synthase (GCS) gene: A preliminary study towards a therapeutic strategy for Gaucher disease and other glycosphingolipid storage diseases.

Small interference RNAs (siRNAs) have recently been used in various experimental settings to silence gene expression. In some of them, chemically synthesized or in vitro transcribed siRNAs have been transfected into cells. In others, siRNAs have been expressed endogenously from siRNA expression vectors. Enzyme replacement and substrate deprivation therapies are currently used to treat Gaucher disease. Although good results have been reported, there are several limitations and side effects that make necessary to search for new alternatives. We present a new approach based on the inhibition of the GCS gene using siRNAs as a potential therapeutic strategy for Gaucher disease. We have designed four siRNAs for the human GCS gene and transfected them into HeLa cells. A clear reduction of GCS RNA levels and enzyme activity was obtained using two of the four siRNAs. Furthermore, a reduction in glucosylceramide synthesis was also observed. Similar results were obtained when plasmids expressing shRNAs (targeting the same sequences) were transfected into the cells. The inhibition of the mouse homolog Ugcg gene was also achieved, using a siRNA that targeted both human and mouse sequences.

Animals↗
PubMed2004

miRNAs on the move: miRNA biogenesis and the RNAi machinery.

Recent advances have led to a more detailed understanding of RNA interference and its role in microRNA biogenesis and function. Primary microRNA transcripts are processed by the RNaseIII nuclease, Drosha, and are exported from the nucleus by Exportin-5. Dicer cleaves microRNAs into their mature forms, which can be incorporated into effector complexes that mediate gene silencing activities. The 3' two-nucleotide overhang structure, a signature of RNaseIII cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway. MicroRNA functional analyses and genetic and biochemical interrogation of components of the pathway are starting to provide a glimpse at the range of biological processes and phenomena regulated by RNA interference.

Animals↗
PubMed2005

RNAi: the nuts and bolts of the RISC machine.

Research on RNA interference and microRNA pathways continues to accelerate. Combinations of biochemical, genetic, and structural approaches are providing new insights into the mechanism by which small, approximately 21 nt long RNAs find their way into the RISC effector complexes and how the RISCs execute their mission of RNA-guided posttranscriptional gene silencing.

Animals↗
PubMed2005

RNAi: RISC gets loaded.

When an siRNA or miRNA proceeds through the RNA-induced silencing complex assembly pathway, only one of the two approximately 21-nucleotide RNA strands survives in the final, active complex. In this issue of Cell, Matranga et al. (2005) and Rand et al. (2005) reveal the fate of the rejected passenger siRNA strand. Additionally, Gregory et al. (2005) define a heterotrimeric complex from humans that appears to execute dsRNA loading, strand selection, and target mRNA cleavage activities.

Animals↗
PubMed2006

A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen.

To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.

Animals↗
PubMed2006

Identification of conserved pathways of DNA-damage response and radiation protection by genome-wide RNAi.

Ionizing radiation is extremely harmful for human cells, and DNA double-strand breaks (DSBs) are considered to be the main cytotoxic lesions induced. Improper processing of DSBs contributes to tumorigenesis, and mutations in DSB response genes underlie several inherited disorders characterized by cancer predisposition. Here, we performed a comprehensive screen for genes that protect animal cells against ionizing radiation. A total of 45 C. elegans genes were identified in a genome-wide RNA interference screen for increased sensitivity to ionizing radiation in germ cells. These genes include orthologs of well-known human cancer predisposition genes as well as novel genes, including human disease genes not previously linked to defective DNA-damage responses. Knockdown of eleven genes also impaired radiation-induced cell-cycle arrest, and seven genes were essential for apoptosis upon exposure to irradiation. The gene set was further clustered on the basis of increased sensitivity to DNA-damaging cancer drugs cisplatin and camptothecin. Almost all genes are conserved across animal phylogeny, and their relevance for humans was directly demonstrated by showing that their knockdown in human cells results in radiation sensitivity, indicating that this set of genes is important for future cancer profiling and drug development.

Animals↗
PubMed2005

A Place for RNAi.

Processing bodies (P bodies) are discrete cytoplasmic foci to which mRNA is routed for degradation. In mammalian cells, they are also associated with miRNA-induced translational silencing and siRNA-induced mRNA degradation. In a recent issue of Molecular Cell, Ding and coworkers described an argonaute-interacting protein that appears to promote the assembly of P bodies in C. elegans (Ding et al., 2005).

Animals↗
PubMed2005

RNAi-mediated knockdown of P-glycoprotein using a transposon-based vector system durably restores imatinib sensitivity in imatinib-resistant CML cell lines.

OBJECTIVE: Resistance to therapeutic drugs is a frequent phenomenon in hematologic malignancies, causing treatment failure in patients with leukemias and lymphomas. Overexpression of the multidrug-resistance gene (MDR-1) and its translational product P-glycoprotein (PgP) represents one mechanism of fatal drug resistance. METHODS: We constructed a nonviral, transposon-based vector system for the stable knockdown of PgP in chronic myeloid leukemia cell lines resistant to imatinib and doxorubicin. RESULTS: Using this strategy, PgP expression was completely knocked down 72 hours after vector inoculation and lasted for several months. Cellular efflux of the PgP substates rhodamine and doxorubicin was abolished. Vector-treated cells were resensitized to imatinib- and doxorubicin-induced cell death. CONCLUSION: Using chronic myeloid leukemia as a model, we show that PgP-mediated resistance to imatinib and anthracyclines can be durably reversed by nonviral, transposon-based knockdown of PgP in malignant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Structural domains in RNAi.

Structural and biochemical studies have begun to elucidate the pathway of RNA silencing that leads to the formation of the RISC complex. The outstanding feature of this pathway is the precise recognition and processing of double-stranded RNA. We present a review of recent structures that illustrate the molecular mechanisms contributing to these two related functions, highlighting models of Drosha, Dicer, and RISC function.

Animals↗