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Results for “RNA Interference”

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PubMed2004

Silencing of chromatin assembly factor 1 in human cells leads to cell death and loss of chromatin assembly during DNA synthesis.

In eukaryotic cells, chromatin serves as the physiological template for gene transcription, DNA replication, and repair. Chromatin assembly factor 1 (CAF-1) is the prime candidate protein to mediate assembly of newly replicated DNA into chromatin. To investigate the physiological role of CAF-1 in vivo, we used RNA interference (RNAi) to silence the 60-kDa subunit of CAF-1 (p60) in human cells. Transfection of a small interfering RNA (siRNA) directed against p60 resulted in efficient silencing of p60 expression within 24 h. This silencing led to an induction of programmed cell death in proliferating but not in quiescent human cells. Concomitantly, proliferating cells lacking p60 accumulated DNA double-strand breaks and increased levels of the phosphorylated histone H2A.X. Nuclear extracts from cells lacking p60 exhibited a 10-fold reduction of nucleosome assembly activity during DNA synthesis, which was restored upon addition of recombinant p60 protein. Nascent chromatin in cell nuclei lacking p60 showed significantly increased nuclease sensitivity, indicating chromatin assembly defects during DNA synthesis in vivo. Collectively, these data identify CAF-1 as an essential factor not only for S-phase-specific chromatin assembly but also for proliferating cell viability.

Animals↗

PubMed2005

Modulation of gene expression by RNAi.

RNA interference (RNAi) is a form of posttranscriptional gene silencing in which the presence within the cell of double-stranded RNA (dsRNA) leads to the specific degradation of mRNA with a complimentary sequence. RNAi is a natural phenomenon that can be exploited as a powerful tool to study gene function by generating gene "knockdowns" in various cell types. RNAi is mediated by short interfering RNAs (siRNAs), which are generated within cells from long dsRNAs. To avoid generalized toxic effects, mammalian cells are transfected directly with 21-23-bp-long siRNAs generated either by chemical synthesis or obtained by a series of enzymatic reactions. The present chapter deals with siRNA design, synthesis, transfection, and readout of efficiency in a mammalian cell culture system. The general principle is illustrated by the functional knockdown of p97/VCP (valosin-containing protein) in HeLa cells using five different siRNA sequences.

Animals↗

PubMed2006

RNAi technology: a revolutionary tool for the colorectal cancer therapeutics.

With the many changes that have taken place in people's diet and lifestyle, colorectal cancer (CRC) has become a global concern. There were approximately 950000 new cases diagnosed and 500000 deaths recorded worldwide in 2000. It is the second most common type of cancer in the Western world, and it is the third most common type of digestive tumor in China. It is reported that the morbidity of CRC is 4.08/100000 for men and 3.30/100000 for women in China. Despite the rate of improvements in surgery, radiotherapy and chemotherapy, the overall five-year survival is around 50%. Therefore, novel treatment need to be developed in order to add to the therapeutic armamentarium. RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing mechanism, which is triggered by double-stranded RNA (dsRNA) and causes degradation of mRNA homologous in sequence to the dsRNA. This new approach has been successfully adopted to inhibit virus replication and tumorigenicity. Recent reports have described DNA vector-based strategies for delivery of small interfering RNA (siRNA) into mammalian cells, further expanding the utility of RNAi. With the development of the RNAi technology and deeper understanding of this field, a promising new modality of treatment appeared, which can be used in combination with the existing therapies. We reviewed the proceedings on the actualities and advancement of RNAi technology for colorectal cancer therapeutics.

Animals↗

PubMed2001

[Inhibition of gene expression by administration of homologous double-stranded RNA in Drosophila melanogaster cell culture].

Specific inhibition of gene expression by exogenous homologous double-stranded RNA (dsRNA) in invertebrates and in the early development of vertebrates is termed RNA interference. Cultured cells were cotransfected with reporter plasmids and dsRNA. The inhibitor effect on reporter gene expression depended on the extent of homology between dsRNA and the target gene. RNA interference was also studied in cells cotransfected with plasmids directing synthesis of sense and antisense RNAs. Production of antisense RNA only slightly inhibited expression of the reporter gene. Simultaneous expression of both sense and antisense RNAs from a special plasmid did not inhibit expression of the reporter construct.

Animals↗

PubMed2004

Generation of an shRNAi expression library against the whole human transcripts.

RNA interference is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, homologous to the gene, and is receiving attention as a powerful tool for reverse genetics in the post-genome era. Throughout our current research to generate an siRNA expression library for the whole human genome, we face many technical difficulties. We present here the strategies for overcoming some of the difficulties, including the development of genetically stable and highly active siRNA expression vectors, the selection procedure of the favorable target sites, and the efficient and low cost procedure for constructing an siRNA expression library. Furthermore, we demonstrate that the screening using the constructed siRNA-expression library indeed works, by evaluating siRNA-expression library against apoptosis-related genes.

Algorithms↗

PubMed2003

RNAi: an ever-growing puzzle.

In recent years, sequence-specific gene silencing has been an area of increasing focus, both because of its interesting biology and because of its power as an experimental tool. A growing understanding of one such phenomenon, RNA interference (RNAi), has provided clues that many homology-dependent gene-silencing mechanisms share a common trigger, double-stranded RNA. Recent findings that RNAi and related pathways are involved not only in the response to exogenous pathogenic and endogenous parasitic nucleic acids but also in basic cellular processes, such as gene regulation and heterochromatin formation, have further fueled interest in this rapidly expanding field.

Animals↗

PubMed2006

Thioredoxin reductase 1 deficiency reverses tumor phenotype and tumorigenicity of lung carcinoma cells.

Dietary selenium has potent cancer prevention activity. Both low molecular weight selenocompounds and selenoproteins are implicated in this effect. Thioredoxin reductase 1 (TR1) is one of the major antioxidant and redox regulators in mammals that supports p53 function and other tumor suppressor activities. However, this selenium-containing oxidoreductase is also overexpressed in many malignant cells and has been proposed as a target for cancer therapy. To further assess the role of TR1 in the malignancy process, we used RNA interference technology to decrease its expression in mouse lung carcinoma (LLC1) cells. Stable transfection of LLC1 cells with a small interfering RNA construct that specifically targets TR1 removal manifested a reversal in the morphology and anchorage-independent growth properties of these cancer cells that made them similar to those of normal cells. The expression of at least two cancer-related protein mRNAs, Hgf and Opn1, were reduced dramatically in the TR1 knockdown cells. Mice injected with the TR1 knockdown showed a dramatic reduction in tumor progression and metastasis compared with those mice injected with the corresponding control vector. In addition, tumors that arose from injected TR1 knockdown cells lost the targeting construct, suggesting that TR1 is essential for tumor growth in mice. These observations provide direct evidence that the reduction of TR1 levels in malignant cells is antitumorigenic and suggest that the enzyme is a prime target for cancer therapy.

Animals↗

PubMed2004

Inhibition of glucocorticoid-induced apoptosis by targeting the major splice variants of BIM mRNA with small interfering RNA and short hairpin RNA.

Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines. To test the hypothesis that Bim facilitates GC-induced apoptosis, we reduced BIM mRNA levels and Bim protein levels by RNA interference in highly GC-sensitive pre-B ALL cells. Reducing Bim proteins by either electroporation of synthetic small interfering RNA (siRNA) duplexes or lentivirus-mediated stable expression of short hairpin RNA inhibited the activation of caspase-3 and increased cell viability following GC exposure. We also observed that the extent of GC resistance correlated with siRNA silencing potency. siRNA duplexes that reduced only BimEL or BimEL and BimL (but not BimS) exhibited less GC resistance than a potent siRNA that silenced all three major isoforms, implying that induction of all three Bim proteins contributes to cell death. Finally, the modulation of GC-induced apoptosis caused by Bim silencing was independent of Bcl-2 expression levels, negating the hypothesis that the ratio of Bim to Bcl-2 regulates apoptosis. These results offer evidence that the induction of Bim by GC is a required event for the complete apoptotic response in pre-B ALL cells.

Acetylcysteine↗

PubMed2006

Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect.

Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGalpha, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGalpha-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC50 value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGalpha/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGalpha/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGalpha/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGalpha-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed.

Biological Transport↗

PubMed2002

Rates of behavior and aging specified by mitochondrial function during development.

To explore the role of mitochondrial activity in the aging process, we have lowered the activity of the electron transport chain and adenosine 5'-triphosphate (ATP) synthase with RNA interference (RNAi) in Caenorhabditis elegans. These perturbations reduced body size and behavioral rates and extended adult life-span. Restoring messenger RNA to near-normal levels during adulthood did not elevate ATP levels and did not correct any of these phenotypes. Conversely, inhibiting respiratory-chain components during adulthood only did not reset behavioral rates and did not affect life-span. Thus, the developing animal appears to contain a regulatory system that monitors mitochondrial activity early in life and, in response, establishes rates of respiration, behavior, and aging that persist during adulthood.

Aging↗

PubMed2006

Gene silencing of myostatin in differentiation of chicken embryonic myoblasts by small interfering RNA.

Myostatin (GDF-8) is known to negatively regulate skeletal muscle mass in myogenesis, but few studies have been conducted on the function of endogenous GDF-8 in primary myoblasts. The present study was performed to assess the function of GDF-8 by RNA interference using primary culture of chicken embryonic myoblasts in which myoblasts were differentiated into myotubes. An active form of small interfering RNA (siRNA-1) targeting GDF-8 mRNA was introduced into myoblasts, and an inactive form of siRNA (siRNA-2) was used as a negative control. GDF-8 transcript level was significantly reduced 24 h after the introduction of siRNA-1 to 25% of the control, whereas a 52-kDa GDF-8 precursor was reduced to 45% of the control at 48 h. However, siRNA-2 did not decrease GDF-8 transcript level. When GDF-8-mediated promoter activity was measured chronologically by means of a pGL(CAGA)(10)-constructed luciferase reporter assay, a concomitant change in activity was initiated after 24 h. The activity rapidly decreased 30 h after siRNA-1 introduction, whereas high activity was maintained at 30-42 h in the control and siRNA-2-treated myoblasts. Myogenic factors such as MyoD and p21, but not myogenin, were altered after 72 h. Cell fusion of the multinucleated myotubes was delayed by the siRNA-1 introduction, and myotubes with aggregated nuclei were shorter and wider. These results strongly suggest that deficiency of GDF-8 delays cell differentiation and causes great alterations in the cellular morphology of chicken embryonic myotubes.

Animals↗

PubMed2006

Small RNAs and non-small cell lung cancer.

Patients with non-small cell lung cancer (NSCLC) are commonly diagnosed with advanced disease and have limited therapeutic options. Experimental treatment approaches including small molecule targeted therapeutics, gene modified tumor vaccines, and viral-based gene therapy have induced tumor regression in a small proportion of patients, suggesting that advanced NSCLC is susceptible to molecular perturbations. RNA interference (RNAi) has generated considerable excitement as a potential cancer therapeutic application. RNAi is the process by which small, double stranded RNA molecules (small interfering RNA, or siRNA) can initiate sequence-specific, post-transcriptional gene silencing (PTGS). Cancer growth inhibition was attained through siRNA-knockdown of unique or overexpressed cancer oncogenetic messages that are relevant to NSCLC pathophysiology. As with other loss-of-function cancer gene therapy approaches, clinical efficacy of siRNA depends largely on the extent of cell target coverage at the locoregional and/or systemic level. Cationic liposomes as well as viral vectors have been used successfully for siRNA delivery. However, viral delivery may have more immediate relevance due to its wider clinical acceptance in the cancer gene therapy arena. We advocate the use of conditional replicative, oncolytic adenovirus for siRNA delivery, which offers potential benefits of restricted and renewable siRNA expression within the tumor microenvironment, and an additive anti-tumor outcome through viral oncolysis and siRNA-mediated oncogene-silencing, which we have demonstrated with the A549 NSCLC cell line. Several oncolytic adenoviral constructs are potentially applicable clinical platforms with proven infectivity and safety, which are feasible also for the delivery of microRNAs (miRNA), a recently discovered group of endogenous, small RNA with PTGS activity that is downregulated in lung cancer.

Carcinoma, Non-Small-Cell Lung↗

PubMed2006

Inhibition of survivin reduces cell proliferation and induces apoptosis in human endometrial cancer.

BACKGROUND: Endometrial cancer is a common gynecologic malignancy among women. The molecular mechanisms involved in the progression of endometrial cancer are unclear, which has hampered the development of an effective treatment. Survivin, a newly identified member of the inhibitor of apoptosis (IAP) family, regulates 2 critical processes in neoplastic transformation: cell proliferation and apoptosis. METHODS: Survivin mRNA and protein expression levels were analyzed in human normal cycling endometrium, atypical endometrium, and endometrial adenocarcinoma by immunohistochemical, reverse-transcriptase polymerase chain reaction (RT-PCR), and Western blot analyses. To study the biological function of survivin in endometrial cancer, RNA interference was applied to knock down survivin expression in the Ishikawa endometrial cancer cell line by recombinant plasmids producing survivin small hairpin RNA. Furthermore, the signal pathway that regulates survivin expression was investigated. RESULTS: Higher levels of survivin mRNA and protein expression were observed in endometrial adenocarcinomas than in atypical or normal endometrium. Immunohistochemical staining revealed that 83.3% (50 of 60) of endometrial adenocarcinoma samples, 55.0% (11 of 20) of atypical endometrium samples, and 25.0% (5 of 20) of normal endometrium samples were positive for survivin protein. Inhibition of survivin by RNA interference reduced cell proliferation and induced apoptosis in Ishikawa cells by down-regulating cyclin D1 and phosphorylated RB and activating caspase-3 and caspase-8. The authors also found that the MAPK pathway was a signal transduction pathway upstream of survivin. Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) upregulated survivin protein expression by activating the MAPK pathway in endometrial cancer cells. CONCLUSIONS: Survivin is an attractive target for endometrial cancer treatment. Growth factors could regulate survivin expression by activating the MAPK pathway.

Adenocarcinoma↗

PubMed2003

Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis.

RNA interference (RNAi) can achieve sequence-selective inactivation of gene expression in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene. Here we explore the potential of RNAi as a therapy for amyotrophic lateral sclerosis (ALS) caused by mutations in the Cu, Zn superoxide dismutase (SOD1) gene. Although the mutant SOD1 is toxic, the wild-type SOD1 performs important functions. Therefore, the ideal therapeutic strategy should be to selectively inhibit the mutant, but not the wild-type SOD1 expression. Because most SOD1 mutations are single nucleotide changes, to selectively silence the mutant requires single-nucleotide specificity. By coupling rational design of small interfering RNAs (siRNAs) with their validation in RNAi reactions in vitro and in vivo, we have identified siRNA sequences with this specificity. A similarly designed sequence, when expressed as small hairpin RNA (shRNA) under the control of an RNA polymerase III (pol III) promoter, retains the single-nucleotide specificity. Thus, RNAi is a promising therapy for ALS and other disorders caused by dominant, gain-of-function gene mutations.

Alleles↗

PubMed2003

Inhibition of apoptosis by Z-VAD-fmk in SMN-depleted S2 cells.

Spinal muscular atrophy is an autosomal recessive motor neuron degenerative disorder, caused by the loss of telomeric copy of the survival motor neuron gene (SMN1). To better understand how motor neurons are targeted in Spinal muscular atrophy patients, it is important to study the role of SMN protein in cell death. In this report, we employed RNA interference (RNAi) to study the loss-of-function of SMN in Drosophila S2 cells. A 601-base pair double-stranded RNA (dsRNA) of Drosophila SMN (dSMN) was used for silencing the dSMN. Our data indicate that dSMN RNAi resulted in more than 90% reduction of both RNA and protein. Further analysis of S2 cells by cell death ELISA and flow cytometry assays revealed that reduction of dSMN expression significantly increased apoptosis. The cell death mediated by SMN depletion is caspase-dependent and specifically due to the activation of the endogenous caspases, DRONC and DRICE. Significantly, the effect of dSMN RNAi was reversed by a peptide caspase inhibitor, Z-VAD-fmk. These results suggest that dSMN is involved in signal pathways of apoptotic cell death in Drosophila. Hence, the model system of reduced SMN expression by RNAi in Drosophila could be exploited for identification of therapeutic targets.

Amino Acid Chloromethyl Ketones↗

PubMed2004

A protein sensor for siRNA asymmetry.

To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two approximately 21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. We show that in Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2. R2D2 binds the siRNA end with the greatest double-stranded character, thereby orienting the heterodimer on the siRNA duplex. Strong R2D2 binding requires a 5'-phosphate on the siRNA strand that is excluded from the RISC. Thus, R2D2 is both a protein sensor for siRNA thermodynamic asymmetry and a licensing factor for entry of authentic siRNAs into the RNAi pathway.

Animals↗

PubMed2004

Effect of inhibition of EBV-encoded latent membrane protein-1 by small interfering RNA on EBV-positive nasopharyngeal carcinoma cell growth.

OBJECTIVE: To evaluate the feasibility of using small interfering RNA (siRNA) for selective inhibiting latent membrane protein-1 (LMP1) expression in Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) cell line C611, and observe the effects of LMP1 gene silencing on the NPC cell growth. METHODS: Four synthesized double-strand siRNA were respectively transfected into NPC cells, using Oligofectamine reagent. The subsequent changes in LMP1 mRNA level were determined by semi-quantitative reverse transcriptase (RT)-PCR. Flow cytometry and MTT assay were employed to examine the alterations in cell cycle and cell proliferation. RESULTS: The most effective sequence was identified among the 4 candidate siRNAs, and its single dose caused nearly 90% loss of LMP1 mRNA in C611 cells, with sustained specific inhibition for 96 h following a re-transfection. LMP1 siRNA treatment resulted in cell cycle arrest at G(0)-G(1) phase, accompanied by a reduction of cell proliferation by 33%, whereas EBV-negative NPC cells appeared unaffected. CONCLUSIONS: EBV-encoded LMP-1 is vulnerable to RNA interference and selective inhibition of LMP1 suppresses the proliferation of EBV-positive NPC cells, a finding that sheds light on the possible use of RNA interference in further investigations of LMP1 and for therapeutic purposes of EBV-related NPC.

Apoptosis↗

PubMed2006

Expression of vector-based small interfering RNA against West Nile virus effectively inhibits virus replication.

RNA interference is one of the effective emerging anti-viral strategies to inhibit virus infection in cells. In this study, a small interfering RNA expressing vector (pSilencer-NS5) targeting the NS5 gene of West Nile virus (WNV) was employed to target and destroy WNV transcripts. Real-time PCR revealed drastic reduction in WNV RNA transcripts in pSilencer-NS5-transfected Vero cells. The virus infectious titre was also significantly reduced by 90% as determined by plaque assays. The resulting decrease in virus replication was shown to be specific since both scrambled and nucleotide(s) mismatch siRNA against WNV NS5 gene did not have any effect on WNV productive yields. Furthermore, Western immunoblot analysis on the expression of viral NS5 and envelope (E) proteins showed significant down-regulation on the expression of viral NS5 and envelope (E) proteins in virus-infected cells that were pre-transfected with pSilencer-NS5. These data clearly supported the notion that the expression of vector-based siRNA against WNV NS5 gene is able to exert its silencing effect on WNV-infected cells without inducing cytotoxicity, hence holding promise in therapeutic treatment of this important emerging infectious disease.

Animals↗