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Results for “RNA interference (RNAi)”

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PubMed2005

Conditional inhibition of cancer cell proliferation by tetracycline-responsive, H1 promoter-driven silencing of PLK1.

RNA interference (RNAi) is a powerful tool for studying gene function. We developed an inducible genetic element for short interfering RNA-mediated gene silencing. This system uses a tetracycline (Tet)-responsive derivative of the H1 promoter and the Tet repressor (TetR) for conditional expression of short hairpin RNA (shRNA) in HeLa cells. Promoter constructs were generated, which contain the Tet operator (TetO) derived from a prokaryotic Tet resistance transposon upstream and/or downstream of the TATA box. To quantify the response of controllable transcription units for shRNA expression, we examined the functional activity of polo-like kinase 1 (PLK1), a key component of mitotic progression, that is overexpressed in many human tumors. Cotransfection of plasmids for the expression of TetR and shRNA/PLK1 under the control of an H1 promoter-variant carrying TetO upstream of the TATA box did not alter PLK1 expression and proliferation properties of HeLa cells in the absence of doxycycline. Addition of the antibiotic led to marked downregulation of endogenous PLK1 accompanied by strong inhibition of cellular proliferation. Our data indicate that an inducible transcription system for shRNAs based on the human H1 promoter could be a versatile tool for controlled gene silencing in vitro.

Cell Cycle Proteins↗
PubMed2002

RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.

Duplexes of 21-nt RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference (RNAi) when introduced into mammalian cells. We show that siRNAs can be synthesized by in vitro transcription with T7 RNA polymerase, providing an economical alternative to chemical synthesis of siRNAs. By using this method, we show that short hairpin siRNAs can function like siRNA duplexes to inhibit gene expression in a sequence-specific manner. Further, we find that hairpin siRNAs or siRNAs expressed from an RNA polymerase III vector based on the mouse U6 RNA promoter can effectively inhibit gene expression in mammalian cells. U6-driven hairpin siRNAs dramatically reduced the expression of a neuron-specific beta-tubulin protein during the neuronal differentiation of mouse P19 cells, demonstrating that this approach should be useful for studies of differentiation and neurogenesis. We also observe that mismatches within hairpin siRNAs can increase the strand selectivity of a hairpin siRNA, which may reduce self-targeting of vectors expressing siRNAs. Use of hairpin siRNA expression vectors for RNAi should provide a rapid and versatile method for assessing gene function in mammalian cells, and may have applications in gene therapy.

Animals↗
PubMed2005

RNAi-directed assembly of heterochromatin in fission yeast.

Heterochromatin is an epigenetically heritable and conserved feature of eukaryotic chromosomes with important roles in chromosome segregation, genome stability, and gene regulation. The formation of heterochromatin involves an ordered array of chromatin changes, including histone deacetylation, histone H3-lysine 9 methylation, and recruitment of histone binding proteins such as Swi6/HP1. Recent discoveries have uncovered a role for the RNA interference (RNAi) pathway in heterochromatin assembly in the fission yeast Schizosaccharomyces pombe and other eukaryotes. Purification of two RNAi complexes, RITS and RDRC, from fission yeast has provided further insight into the mechanism of RNAi-mediated heterochromatin assembly. These discoveries have given rise to a model in which small interfering RNA molecules act as specificity factors that initiate epigenetic chromatin modifications and double strand RNA synthesis at specific chromosome regions.

Animals↗
PubMed2005

TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing.

Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. In this work, we describe the identification of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) as a protein partner of human Dicer. We show that TRBP is required for optimal RNA silencing mediated by siRNAs and endogenous miRNAs, and that it facilitates cleavage of pre-miRNA in vitro. TRBP had previously been assigned several functions, including inhibition of the interferon-induced double-stranded RNA-regulated protein kinase PKR and modulation of HIV-1 gene expression by association with TAR. The TRBP-Dicer interaction shown raises interesting questions about the potential interplay between RNAi and interferon-PKR pathways.

Cell Line↗
PubMed2006

RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection.

Mass sequencing of cDNA libraries from salivary glands of triatomines has resulted in the identification of many novel genes of unknown function. The aim of the present work was to develop a functional RNA interference (RNAi) technique for Rhodnius prolixus, which could be widely used for functional genomics studies in triatomine bugs. To this end, we investigated whether double-stranded RNA (dsRNA) can inhibit gene expression of R. prolixus salivary nitrophorin 2 (NP2) and what impact this might have on anticoagulant and apyrase activity in the saliva. dsRNA was introduced by two injections or by ingestion. RT-PCR of the salivary glands showed that injections of 15 microg of NP2 dsRNA in fourth-instar nymphs reduced gene expression by 75+/-14% and that feeding 1 microg/microL of NP2 dsRNA into second-instar nymphs (approx. 13 microg in total) reduced gene expression by 42+/-10%. Phenotype analysis showed that saliva of normal bugs prolonged plasma coagulation by about four-fold when compared to saliva of knockdown bugs. These results and the light color of the salivary gland content from some insects are consistent with the knockdown findings. The findings suggest that RNAi will prove a highly valuable functional genomics technique in triatomine bugs. The finding that feeding dsRNA can induce knockdown is novel for insects.

Animals↗
PubMed2005

The future of HIV infection: gene therapy and RNA interference.

The description of the mechanism of RNA interference (RNAi) has generated enormous interest in the biomedical field. A previously unrecognized pathway in which small interfering, 21 to 23 mer, double-stranded RNA (siRNA) mediates sequence-specific degradation of mRNA is becoming one the most useful techniques in cell biology and genetics research. Based on the potency, specificity and physiology of RNAi to silence gene expression, much is expected from its use as a therapeutic tool. The first evidence of RNAi as a suppressor of HIV replication has already been reported, thus providing a new impetus to the development of molecular or gene therapy approaches to HIV infection.

Adult↗
PubMed2004

Suppression of Epstein-Barr virus-encoded latent membrane protein-1 by RNA interference inhibits the metastatic potential of nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a highly metastatic tumor characterized by close association with EBV. Of the EBV-encoded products, latent membrane protein-1 (LMP-1) is thought to be the only oncoprotein playing an essential role in cell transformation as well as tumor metastasis. In this study, we tested the effect of suppressing LMP-1 by RNA interference (RNAi) on the proliferative and metastatic potentials of an EBV-positive NPC cell line, C666. We showed that stably suppressing LMP-1 by short hairpin RNA (shRNA) plasmid significantly altered cell motility, substratum adhesion, and transmembrane invasion ability. However, it has little effect on the rate of cell growth and cell cycle control. These results demonstrated the effectiveness of RNAi in suppressing LMP-1, supporting an important role of LMP-1 in NPC metastasis, and suggested a potential application of RNAi-mediated therapeutic strategy for EBV-related NPC.

Cell Adhesion↗
PubMed2005

Analysis of short interfering RNA function in RNA interference by using Drosophila embryo extracts and schneider cells.

The realization that short double-stranded RNA (dsRNAs) 21-25 bp in length represent the basis for posttranscriptional gene silencing (PTGS) in plants, quelling in N. crassa, and RNA interference (RNAi) in C. elegans and Drosophila has given insight into one of the most evolutionarily conserved pathways in eukaryotes. dsRNA that arises due to viral infection, transposon mobilization, random insertion of transgenes near active promoters, transcripts from repetitive elements in the genome, or introduction of exogenous dsRNA directly is processed by one of the RNase III-related enzymes, known as the Dicers, to produce 21- to 25-bp short dsRNAs or short interfering RNAs (siRNAs) that target the degradation of the cognate RNA sequence (Denli and Hannon, 2003; Hannon, 2002; Plasterk, 2002). Proteins in the RNAi pathway and siRNA-like RNAs have also been recently demonstrated to play a role in the formation and maintenance of heterochromatin in S. pombe as well as in transgene-induced PTGS in Drosophila (Hall et al., 2002; Pal-Bhadra et al., 2004; Volpe et al., 2002). An understanding of siRNA function in these crucial regulatory pathways requires biochemical approaches to study siRNAs and their role in gene silencing as well as the formation and maintenance of heterochromatin. This chapter describes simple methods for using Drosophila embryo extracts and cultured insect cells to study siRNA function in the RNAi pathway in vivo and in vitro. We describe the most recent protocols for the preparation and use of Drosophila embryo extracts used in gene targeting studies. We present methods we have used to assay siRNA function in Drosophila embryo extracts and in cultured SL2 cells that demonstrate a combined role for siRNAs and RNA-dependent RNA polymerase (RdRp) activity in Drosophila RNAi.

Animals↗
PubMed2004

Biochemical dissection of RNA silencing in plants.

Although RNA silencing was first discovered in plants, thus far it has been studied biochemically only in animals, where it is known as RNA interference (RNAi). In animals, two components of the RNAi pathway have been identified: Dicer, a multidomain RNase III that converts long double-stranded RNA (dsRNA) into small interfering RNA (siRNA) and the RNA-induced silencing complex (RISC), as siRNA-containing protein-RNA complex that targets complementary mRNA for destruction. We have developed methods for the biochemical dissection of plant RNA silencing. In this chapter, we describe in detail how to use wheat germ extract to study two distinct Dicer-like activities, RNA-dependent RNA polymerase (RdRP), and endogenous microRNA-programmed RISC activities. These comprehensive protocols should prove useful in the further dissection of the plant RNA silencing pathway, as well as for the validation of the predicted targets of endogenous plant microRNAs.

Animals↗
PubMed2005

[Effects of inhibiting SDF-1 expression by RNA interference on adhesion and drug sensitivity of Jurkat cells co-cultured with bone marrow stromal cells].

OBJECTIVE: To observe the effects of inhibiting stromal cell derived factor-1 (SDF-1) expression by RNA interference (RNAi) on adhesion and drug sensitivity of Jurkat cells co-cultured with bone marrow stromal cells. METHODS: SDF-1 specific short hairpin RNA (shRNA) expressing plasmid was transferred into cultured human acute leukemic bone marrow stromal cells, positive clones were isolated by screening G418 resistance (Group A) , SDF-1 protein level in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The adhesion rates to bone marrow stromal cells layer and the drug sensitivity to doxorubicin of co-cultured Jurkat cells were detected by cell counting and MTT assay, respectively. The un-transfected bone marrow stromal cells of acute leukemia patient (Group B) or normal subject (Group C) were taken as control. RESULTS: The level of secreted SDF-1 protein (pg/10(5) cells/week) in the supernatants of Group A, B and C were 1920 +/- 205, 12,370 +/- 1355 and 6620 +/- 770, respectively. Of co-cultured Jurkat cells in Group A, B and C, the adhesion rates after 24 h co-culturing were (28.8 +/- 2.6)%, (57.4 +/- 3.8)% and (45.2 +/- 4.0)%, respectively, and the IC50 values of doxorubicin were 585, 6162 and 1758 nmol/L, respectively. CONCLUSION: Down-regulating SDF-1 expression of bone marrow stromal cells by RNAi reduces adhesion rates and enhances drug sensitivity to doxorubicin of their co-cultured Jurkat cells.

Bone Marrow Cells↗
PubMed2006

Selective blocking of clathrin-mediated endocytosis by RNA interference: epsin as target protein.

Epsin is an essential accessory protein exclusively implicated in clathrin-mediated endocytosis and therefore an ideal target to study involvement of this entry route in the uptake of bioligands. The technique of RNA interference (RNAi) was exploited to generate a cell line constitutively silencing epsin expression in a sequence-specific manner In these Caco-2(eps-) cells, quantitative reverse transcription PCR (RT-PCR) revealed a severe depletion of the epsin messenger RNA (mRNA) level in cells, reaching a factor > 10(6). The reduction at the mRNA level in the Caco-2(eps-) cells was paralleled by a decrease of 75% at the protein level. In order to evaluate transfection effects at the functional level, uptake of transferrin and epidermal growth factor (EGF) in transfected Caco-2(eps-) and control cells was evaluated. In control cells, respectively, approximately 72% of transferrin and approximately 66% of EGF were internalized, whereas in Caco-2(eps-) cells only approximately 25% of transferrin and approximately 34% of EGF was taken up, confirming that in the transfected cells, endocytosis via coated pits was prominently compromised. The reduced uptake was not the result of an inhibition of transferrin recycling. The effects of direct treatment with chlorpromazine on Caco-2 cells, also monitored from the degree of transferrin internalization, were compared with those elicited by RNAi.

Adaptor Proteins, Vesicular Transport↗
PubMed2002

Double-stranded RNA-mediated gene silencing of cysteine proteases (falcipain-1 and -2) of Plasmodium falciparum.

Malaria remains a public health problem of enormous magnitude, affecting over 500 million people every year. Lack of success in the past in the development of new drug/vaccines has mainly been attributed to poor understanding of the functions of different parasite proteins. Recently, RNA interference (RNAi) has emerged as a simple and incisive technique to study gene functions in a variety of organisms. In this study, we report the results of RNAi by double-stranded RNA of cysteine protease genes (falcipain-1 and -2) in the malaria parasite, Plasmodium falciparum. Using RNAi directed towards falcipain genes, we demonstrate that blocking the expression of these genes results in severe morphological abnormalities in parasites, inhibition of parasite growth in vitro and substantial accumulation of haemoglobin in the parasite. The inhibitory effects produced by falcipain double-stranded (ds)RNAs are reminiscent of the effects observed upon administering E-64, a cysteine protease inhibitor. The parasites treated with falcipain's dsRNAs also show marked reduction in the levels of corresponding endogenous falcipain mRNAs. We also demonstrate that dsRNAs of falcipains are broken into short interference RNAs approximately 25 nucleotides in size, a characteristic of RNAi, which in turn activates sequence-specific nuclease activity in the malaria parasites. These results thus provide more evidence for the existence of RNAi in P. falciparum and also suggest possibilities for using RNAi as an effective tool to determine the functions of the genes identified from the P. falciparum genome sequencing project.

Animals↗
PubMed2006

[Influence of survivin gene repression by RNA interference on the radiosensitivity and chemosensitivity to cisplatin of cervical cancer cell HeLa].

OBJECTIVE: To observe the effect of survivin gene repression by RNA interference (RNAi) on the radiosensitivity and the chemosensitivity to cisplatin of cervical cancer cell HeLa. METHODS: The recombined eukaryotic expression plasmid pSilencer2.1-s2 containing human survivin gene small interference RNA (siRNA) was transfected into human cervical cancer cell line HeLa by using lipofectamine 2000. The expression of survivin gene mRNA and its protein was detected by semi-quantitative RT-PCR and western blot respectively. The changes of caspase-3 activity was assessed by kinase activity test. Cell apoptosis was examined by flow cytometry. The changes of cell radiosensitivity was observed by plate clone formation assay. Cell viability was examined by methyl thiazolyl tetrazolium (MTT) assay and 50% inhibitive concentration (IC(50)) of cisplatin was examined also. RESULTS: Compared with the cells which were transfected with negative control plasmid (HeLa-NC), pure plasmid (HeLa-U6 neo) and un-transfected cells (HeLa), the expression level of survivin gene mRNA and protein declined evidently in the cells transfected with pSilencer2.1-s2 plasmid (HeLa-s2), the expression inhibitory rates were (62.8 +/- 0.3)% and (60.1 +/- 0.5)%; the caspase-3 activity was enhanced and A(405) was 1.261 +/- 0.043 (P < 0.05); the apoptotic rate was increased obviously (29.23 +/- 1.41)% (P < 0.05). At the same dose of radiation, clone formation rate declined significantly (P < 0.05); at the same concentration of cisplatin, cell viability declined sharply and the IC(50) of cisplatin was (0.873 +/- 0.021) microg/ml (P < 0.05). CONCLUSION: Survivin gene repression by RNAi can enhance caspase-3 activity, induce cell apoptosis, significantly increase the radiosensitivity and chemosensitivity to cisplatin in human cervical cancer cell line HeLa.

Antineoplastic Agents↗
PubMed2006

Cellular phenotyping by RNAi.

A systematic characterization of genes with unknown function is a key challenge after the sequencing of the human genome and the genomes of many model organisms. High-throughput RNA-interference (RNAi) screenings have become a widely used approach in invertebrate model organisms and also promise to revolutionize cell biology in mammals. Genome-wide RNAi screens in Caenorhabditis elegans and Drosophila, and in a smaller scale in mammalian cells have proven to be a valuable and successful method for the dissection of diverse biological processes. A number of RNAi libraries have become available that rely on different technologies, such as long double-stranded (ds) RNAs, in vitro diced short-interfering (si) RNAs, synthetic siRNAs and short-hairpin (sh) RNAs, which all have specific advantages and disadvantages. In addition, progress in screening technologies and data analysis allows the adaptation of screening methods to analyse more complex cellular processes. This review will summarize strategies in combining genome-scale RNAi libraries, high-throughput screening technologies, integrated high-content data analysis and will discuss future challenges.

Animals↗
PubMed2004

siRNA-mediated inhibition of HBV replication and expression.

AIM: RNA interference (RNAi) is a newly discovered phenomenon provoked by dsRNA. The dsRNA is initially cleaved by Dicer into 21-23 nt small interfering RNA (siRNA) and can then specifically target homologous mRNA for degradation by cellular ribonucleases. RNAi has been successfully utilized to down-regulate the endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this study, we investigated whether vector-based siRNA promoted by U6 (pSilencer1.0-U6) could efficiently inhibit HBV replication in cell culture. METHODS: pSilencer vectors with inserts targeting on different regions of HBV genome were constructed. These plasmids were co-transfected with pHBV3.8 into Huh-7 cells via lipofection and viral antigens were measured by ELISA. Viral RNA was analyzed by Northern blot. The mRNA of MxA and 2'-5'OAS was reverse transcribed and quantified by real-time PCR. RESULTS: Vector-based siRNA could potently reduce hepatitis B virus antigen expression in transient replicative cell culture. Furthermore, Northern blot analysis showed that viral RNA was effectively degraded, thus eliminating the messengers for protein expression as well as template for reverse transcription. Real-time PCR analysis of cellular MxA and 2'-5'OAS gene expression revealed that vector-based siRNA did not provoke the interferon pathway which reassured the specificity of the vector-based RNA interference technique. CONCLUSION: Our results indicate that RNA interference may be a potential tool to control HBV infection.

Blotting, Northern↗
PubMed2003

RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum.

RNA interference (RNAi), a gene silencing process, has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned putative Amblyomma americanum histamine binding protein (HBP) to test the significance of using this methodology in the assessment of the function and importance of gene products in ectoparasitic ticks. The female salivary glands incubated in vitro with HBP dsRNA had a significantly lower histamine binding ability. In addition, the injection of HBP dsRNA into the unfed females led both to a reduced histamine binding ability in the isolated salivary glands and to an aberrant tick feeding pattern or host response. Molecular data demonstrated less expression of the HBP mRNA in the RNAi group. Taken together, these results suggest that RNAi might be an important tool for assessing the significance of tick salivary gland secreted proteins modulating responses at the tick-host interface.

Animals↗
PubMed2004

Gene silencing of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis by RNA interference.

RNA interference (RNAi) has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned Haemaphysalis longicornis serine proteinase (HlSP) gene to test the importance of the function of the HlSP gene product during blood-feeding. Growth of unfed ticks treated with HlSP dsRNA was significantly inhibited compared to that of PBS-treated ticks. This inhibition was supported by the level of HlSP mRNA. HlSP may play a crucial role for blood-feeding in these ticks. This is the first report on gene silencing of a functional serine proteinase in hard ticks.

Animals↗
PubMed2005

Inhibition of multiple gene expression and virus replication of HBV by stable RNA interference in 2.2.15 cells.

BACKGROUND/AIMS: Hepatitis B virus (HBV) infection is a world-wide health problem. Recent studies have demonstrated the efficacy of RNA interference (RNAi) against HBV replication at cell culture and animal levels using transient transfection. The present study was to determine whether the stable transfection of short hairpin RNA (shRNA)-producing vector could achieve potent and sustained inhibition of the HBV replication in 2.2.15 cells. METHODS: shRNA-producing vector against HBV and the empty vector were stably transfected into the 2.2.15 cells respectively. A series of experiments were performed in the producing stable lines to determine the changes of viral protein expression and replication. RESULTS: The HBV protein expression and viral replication were suppressed dramatically and stably by the integrated shRNA-producing vectors. Most importantly, this suppression effect persists after 30 passages. CONCLUSIONS: Our data provided the possibility of continuous and stable inhibition of HBV protein expression and replication in patients using RNAi, suggesting a potential clinical application of this novel approach. Furthermore, the established stable transfected cell lines provided a good platform for understanding the mechanism of anti-HBV by RNAi.

Blotting, Northern↗