PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “RNA interference”

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 973 records · Page 54Linked to original sources
PubMed2003

RNA interference in the moss Physcomitrella patens.

The moss Physcomitrella patens performs efficient homologous recombination, which allows for the study of individual gene function by generating gene disruptions. Yet, if the gene of study is essential, gene disruptions cannot be isolated in the predominantly haploid P. patens. Additionally, disruption of a gene does not always generate observable phenotypes due to redundant functions from related genes. However, RNA interference (RNAi) can provide mutants for both of these situations. We show that RNAi disrupts gene expression in P. patens, adding a significant tool for the study of plant gene function. To assay for RNAi in moss, we constructed a line (NLS-4) expressing a nuclearly localized green fluorescent protein (GFP):beta-glucuronidase (GUS) fusion reporter protein. We targeted the reporter protein with two RNAi constructs, GUS-RNAi and GFP-RNAi, expressed transiently by particle bombardment. Transformed protonemal cells are marked by cobombardment with dsRed2, which diffuses between the nucleus and cytoplasm. Cells transformed with control constructs have nuclear/cytoplasmic red fluorescence and nuclear green fluorescence. In cells transformed with GUS-RNAi or GFP-RNAi constructs, the nuclear green fluorescence was reduced on average 9-fold as soon as 48 h after transformation. Moreover, isolated lines of NLS-4 stably transformed with GUS-RNAi construct have silenced nuclear GFP, indicating that RNAi is propagated stably. Thus, RNAi adds a powerful tool for functional analysis of plant genes in moss.

Bryopsida↗
PubMed2002

Reducing hypothalamic AGRP by RNA interference increases metabolic rate and decreases body weight without influencing food intake.

BACKGROUND: Several lines of evidence strongly suggest that agouti-related peptide (AGRP) plays a key role in the regulation of metabolic function but ablation of the AGRP gene has no apparent effect on metabolic function. Since specific pharmacological antagonists of AGRP do not presently exist, we assessed if reduction of hypothalamic AGRP mRNA by RNA interference (RNAI) would influence metabolic function, an outcome suggesting that pharmacological antagonists might constitute useful reagents to treat obesity. RESULTS: The RNAI protocol specifically reduced hypothalamic expression of AGRP mRNA by 50% and resulted in reduction of AGRP peptide immunoreactivity. Physiologically, the reduction in AGRP levels was associated with increased metabolic rate and reduced body weight without changes in food intake. CONCLUSION: AGRP can function to increase body weight and reduce metabolic rate without influencing food intake. The present study demonstrates that RNAI protocols can be used to assess physiological function of neuronal genes in vivo.

Agouti-Related Protein↗
PubMed2003

RNA interference: from an ancient mechanism to a state of the art therapeutic application?

Now that the sequencing of many genomes has been completed, the basic challenges are finding the genes and predicting their functions. Up until now, a large information gap has existed between the knowledge of genome sequence and our knowledge of protein function. The assessment of gene function may be performed using the tools of reverse genetics, including knock-out mice, antisense oligomers, aptamers, and ribozymes. These approaches have been superseded by RNA interference (RNAi), which exhibits much more potency for the investigation of protein function than the techniques listed above. As already known some years ago, RNAi is based on an ancient anti-viral defense mechanism in lower eukaryotes. It is induced by double-stranded RNA and its processing to 21-23 nt small interfering RNAs (siRNAs), which cause the degradation of homologous endogenous mRNA. The way RNAi works has still to be determined, but it already serves as a first-choice approach to generate loss-of-function phenotypes among a broad variety of eukaryotic species, such as nematodes, flies, plants, fungi and mammals. RNAi also represents an extremely powerful tool, becoming a therapeutic approach to curing infectious diseases originated by viral or parasitic invasion. In this review we present the current view of how RNAi works in different eukaryotic species and its high potential for functional genomics and in rational drug design.

Animals↗
PubMed2006

Downregulation of human Cdc6 protein using a lentivirus RNA interference expression vector.

Eukaryotic CDC6 gene function is required for the initiation of DNA replication and is a key regulatory protein during cell cycle progression. The human CDC6 gene is not expressed in most normal tissues, in contrast with its marked expression in proliferating cancer cells. An effective way to explore the gene functions of CDC6 is to knock-down the CDC6 messenger RNA (mRNA) and examine the phenotypic consequences. In this chapter, we describe the construction of a lentivirus vector to express a CDC6 DNA segment. The transcript is able to fold by itself because the sense and antisense regions are complementary. There is a 9-nucleotide (nt) loop region allowing for the short hairpin RNA (shRNA) to form. Cellular ribonucleases process the shRNA into a functional short interfering RNA (siRNA). Down-regulation of Cdc6 protein is confirmed by Western blots.

Cell Cycle Proteins↗
PubMed2005

RNA interference: new drugs on the horizon.

By wrapping chemically modified siRNA in a specialized liposome the in vivo potency and duration of action of siRNA targeted against hepatitis B virus has greatly increased.

Genetic Therapy↗
PubMed2005

Down-regulation of CT120A by RNA interference suppresses lung cancer cells growth and sensitizes to ultraviolet-induced apoptosis.

CT120A gene was isolated from chromosome 17p13.3 by using positional cloning and RACE by our laboratory. Here, we reported the evidence that CT120A protein was a potential molecular target for treatment of lung cancers. CT120A was overexpressed in 15 cases of the 16 primary lung cancer specimens. Knockdown of CT120A by small hairpin RNA in the human lung adenocarcinoma cell line SPC-A-1 cells resulted in a reduced cell growth rate in vitro and decrease of the capacity for anchorage-independent growth and tumorigenicity in nude mice. The suppression of CT120A expression also sensitized cells to ultraviolet-induced apoptosis. Atlas cDNA expression array revealed that the expressions of several apoptosis- and growth-associated genes were altered underlying the molecular mechanisms of these cell biological behaviors. These results suggested that CT120A was a novel lung cancer-related gene and CT120A might be a potential target for therapeutic anti-cancer drugs.

Adenocarcinoma↗
PubMed2006

Silencing the activity and proliferative properties of the human EagI Potassium Channel by RNA Interference.

EagI potassium channels are natively expressed in the mammalian brain as well as in many cancer cell lines and tumor tissues. The role of EagI in malignant transformation has been suggested by several experiments, but the lack of specific EagI inhibitors has made it difficult to examine the influence of the channel on oncogenesis and its potential as a therapeutic target. We have used short interfering RNA to test the effects of EagI reduction on the behavior of tumor cells in vitro. By generating and optimizing an EagI-specific short interfering RNA system, we were able to study the effects of EagI depletion on several cancer cell lines that endogenously express this protein. We show here that our short interfering RNA sequences act specifically on EagI, reproducibly induce a significant decrease in the proliferation of tumor cell lines, and do not trigger any observable nonspecific responses.

Cell Line, Tumor↗
PubMed2004

RNA interference as a potential tool in the treatment of leukaemia.

Leukaemias are often characterised by nonrandom chromosomal translocations that, at the molecular level, induce the activation of specific oncogenes or create novel chimeric genes. They have frequently been regarded as optimal targets for gene silencing approaches, as these single abnormalities may directly initiate or maintain the malignant process. Since the ground-breaking discovery that double-stranded RNA molecules 21 - 23 nucleotides in length, named small interfering RNAs (siRNAs), are able to elicit gene-specific inhibition also in mammalian cells, the interest of the scientific community has rapidly been drawn to the potential of these siRNAs for targeting oncogenic fusion genes in leukaemic cells. There has been a flurry of reports describing overexpressed or mutated genes that may also serve as attractive targets for therapeutic intervention by RNA silencing methods. Although this approach seems to be relatively straightforward, many problems remain to be solved before siRNAs may become clinically implemented as 'leukaemia drugs'. Difficulties in delivering siRNAs into the leukaemic cell, inefficient target mRNA cleavage, prolonged protein half-life in cancer cells, nonspecific side effects caused by targeting other genes than those originally thought, immunological reactions of the host organism against the siRNAs, such as interferon responses, or even acquired resistance mechanisms, such as escape mutants, should be overcome. This paper reviews the current knowledge regarding the use of siRNAs, either chemically synthesised or intracellular-generated via specialised expression constructs, in order to suppress the falsely activated oncogenes in haematopoietic malignancies.

Gene Targeting↗
PubMed2006

Study of claudin function by RNA interference.

Claudins are tight junction proteins that play a key selectivity role in the paracellular conductance of ions. Numerous studies of claudin function have been carried out using the overexpression strategy to add new claudin channels to an existing paracellular protein background. Here, we report the systematic knockdown of endogenous claudin gene expression in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells using small interfering RNA against claudins 1-4 and 7. In MDCK cells (showing cation selectivity), claudins 2, 4, and 7 are powerful effectors of paracellular Na+ permeation. Removal of claudin-2 depressed the permeation of Na+ and resulted in the loss of cation selectivity. Loss of claudin-4 or -7 expression elevated the permeation of Na+ and enhanced the proclivity of the tight junction for cations. On the other hand, LLC-PK1 cells express little endogenous claudin-2 and show anion selectivity. In LLC-PK1 cells, claudin-4 and -7 are powerful effectors of paracellular Cl- permeation. Knockdown of claudin-4 or -7 expression depressed the permeation of Cl- and caused the tight junction to lose the anion selectivity. In conclusion, claudin-2 functions as a paracellular channel to Na+ to increase the cation selectivity of the tight junction; claudin-4 and -7 function either as paracellular barriers to Na+ or as paracellular channels to Cl-, depending upon the cellular background, to decrease the cation selectivity of the tight junction.

Animals↗
PubMed2006

A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action.

BACKGROUND: All archaeal and many bacterial genomes contain Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR) and variable arrays of the CRISPR-associated (cas) genes that have been previously implicated in a novel form of DNA repair on the basis of comparative analysis of their protein product sequences. However, the proximity of CRISPR and cas genes strongly suggests that they have related functions which is hard to reconcile with the repair hypothesis. RESULTS: The protein sequences of the numerous cas gene products were classified into approximately 25 distinct protein families; several new functional and structural predictions are described. Comparative-genomic analysis of CRISPR and cas genes leads to the hypothesis that the CRISPR-Cas system (CASS) is a mechanism of defense against invading phages and plasmids that functions analogously to the eukaryotic RNA interference (RNAi) systems. Specific functional analogies are drawn between several components of CASS and proteins involved in eukaryotic RNAi, including the double-stranded RNA-specific helicase-nuclease (dicer), the endonuclease cleaving target mRNAs (slicer), and the RNA-dependent RNA polymerase. However, none of the CASS components is orthologous to its apparent eukaryotic functional counterpart. It is proposed that unique inserts of CRISPR, some of which are homologous to fragments of bacteriophage and plasmid genes, function as prokaryotic siRNAs (psiRNA), by base-pairing with the target mRNAs and promoting their degradation or translation shutdown. Specific hypothetical schemes are developed for the functioning of the predicted prokaryotic siRNA system and for the formation of new CRISPR units with unique inserts encoding psiRNA conferring immunity to the respective newly encountered phages or plasmids. The unique inserts in CRISPR show virtually no similarity even between closely related bacterial strains which suggests their rapid turnover, on evolutionary scale. Corollaries of this finding are that, even among closely related prokaryotes, the most commonly encountered phages and plasmids are different and/or that the dominant phages and plasmids turn over rapidly. CONCLUSION: We proposed previously that Cas proteins comprise a novel DNA repair system. The association of the cas genes with CRISPR and, especially, the presence, in CRISPR units, of unique inserts homologous to phage and plasmid genes make us abandon this hypothesis. It appears most likely that CASS is a prokaryotic system of defense against phages and plasmids that functions via the RNAi mechanism. The functioning of this system seems to involve integration of fragments of foreign genes into archaeal and bacterial chromosomes yielding heritable immunity to the respective agents. However, it appears that this inheritance is extremely unstable on the evolutionary scale such that the repertoires of unique psiRNAs are completely replaced even in closely related prokaryotes, presumably, in response to rapidly changing repertoires of dominant phages and plasmids.

Journal Article↗
PubMed2003

RNA interference targeting focal adhesion kinase enhances pancreatic adenocarcinoma gemcitabine chemosensitivity.

Focal adhesion kinase (FAK) is an important regulator of cellular signaling, migration, apoptosis, and cell cycle progression. We tested the hypothesis that FAK is a determinant of gemcitabine chemoresistance in pancreatic adenocarcinoma cells and examined the effect of inhibiting FAK expression on gemcitabine-induced cytotoxicity in vitro and in vivo. FAK expression was quantified by Western and Northern blots. Expression of FAK was suppressed using small interfering RNA (siRNA). Gemcitabine-induced cytotoxicity was quantified and apoptosis was characterized. Akt activity was determined by in vitro kinase assay. We assessed the therapeutic applicability of FAK siRNA in a nude mouse orthotopic xenograft model. While not affecting cellular proliferation or apoptosis in the absence of gemcitabine, FAK siRNA potentiated gemcitabine-induced cytotoxicity in vitro and in vivo. FAK siRNA treatment suppressed Akt activity, which may contribute to its chemosensitizing effect.

Adenocarcinoma↗
PubMed2004

Targeting Alzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles.

Tau and amyloid precursor protein (APP) are key proteins in the pathogenesis of sporadic and inherited Alzheimer's disease. Thus, developing ways to inhibit production of these proteins is of great research and therapeutic interest. The selective silencing of mutant alleles, moreover, represents an attractive strategy for treating inherited dementias and other dominantly inherited disorders. Here, using tau and APP as model targets, we describe an efficient method for producing small interfering RNA (siRNA) against essentially any targeted region of a gene. We then use this approach to develop siRNAs that display optimal allele-specific silencing against a well-characterized tau mutation (V337M) and the most widely studied APP mutation (APPsw). The allele-specific RNA duplexes identified by this method then served as templates for constructing short hairpin RNA (shRNA) plasmids that successfully silenced mutant tau or APP alleles. These plasmids should prove useful in experimental and therapeutic studies of Alzheimer's disease. Our results suggest guiding principles for the production of allele-specific siRNA, and the general method described here should facilitate the production of gene-specific siRNAs.

Alleles↗
PubMed2005

Elucidating the role of H/ACA-like RNAs in trans-splicing and rRNA processing via RNA interference silencing of the Trypanosoma brucei CBF5 pseudouridine synthase.

Most pseudouridinylation in eukaryotic rRNA and small nuclear RNAs is guided by H/ACA small nucleolar RNAs. In this study, the Trypanosoma brucei pseudouridine synthase, Cbf5p, a snoRNP protein, was identified and silenced by RNAi. Depletion of this protein destabilized all small nucleolar RNAs of the H/ACA-like family. Following silencing, defects in rRNA processing, such as accumulation of precursors and inhibition of cleavages to generate the mature rRNA, were observed. snR30, an H/ACA RNA involved in rRNA maturation, was identified based on prototypical conserved domains characteristic of this RNA in other eukaryotes. The silencing of CBF5 also eliminated the spliced leader-associated (SLA1) RNA that directs pseudouridylation on the spliced leader RNA (SL RNA), which is the substrate for the trans-splicing reaction. Surprisingly, the depletion of Cbf5p not only eliminated the pseudouridine on the SL RNA but also abolished capping at the fourth cap-4 nucleotide. As a result of defects in the SL RNA and decreased modification on the U small nuclear RNA, trans-splicing was inhibited at the first step of the reaction, providing evidence for the essential role of H/ACA RNAs and the modifications they guide on trans-splicing.

Alternative Splicing↗