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PubMed2006

Application of advanced technologies in ageing research.

Several technologies that emerged in the post-genomic era have been particularly useful in dissecting the molecular mechanisms of complex biological processes through the systems approach. Here, we review how three of these technologies, namely transcriptional profiling, large-scale RNA interference (RNAi) and genome-wide location analysis of protein-DNA interactions, have been used in the study of ageing in metazoans. We also highlight recent developments of these three technologies and how these developments are applicable to ageing research.

Aging↗

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

Silencing of HIV-1 subtype C primary isolates by expressed small hairpin RNAs targeted to gag.

Discovery of sequence-specific silencing by activating the RNA interference (RNAi) pathway has led to exciting new strategies for treating infection with human immunodeficiency virus type 1 (HIV-1). Of the HIV-1 subtypes, C is especially common in areas of the world that are worst affected. Although prone to mutation, genome plasticity of this subtype is limited in functionally important regions. We identified conserved sequences within the HIV-1 subtype C gag open reading frame and assessed whether they are suitable targets for inhibition of viral replication by RNA Pol III-driven small hairpin RNAs (shRNAs). Initially, the efficacy of each of a panel of 10 shRNAs against HIV-1 was determined using a reporter assay. shRNAs A and B, which targeted the 5 end of gag, were most effective and were used to assess inhibition of replication in cultured cells of two R5 isolates (Du151 and Du422) and one X4 virus (SW7). These shRNAs diminished intracellular HIV-1 gag RNA and HIV-1 protein concentrations as well as p24 secretion by up to 80% without inducing an interferon response. However, shRNA-mediated knockdown efficacy against each of these viral isolates varied slightly. These data show successful activation of RNAi to inhibit the replication of biologically distinct HIV-1 subtype C isolates. The effector shRNAs described here are potential candidates for gene therapy applications against the most common global subtype of HIV-1.

Cell Line↗

PubMed2005

Construction of influenza virus siRNA expression vectors and their inhibitory effects on multiplication of influenza virus.

Three plasmid constructs were prepared that express small interfering RNAs (siRNAs) targeted to sequences encoding the ribonucleoprotein member, nucleoprotein (NP) and/or PA, of influenza virus genome. The antiviral properties of siRNAs against the H5N1 strain of influenza virus were studied by evaluating their capacity to silence expression of target genes as well as their effect on influenza virus-induced apoptosis in Madin-Darby canine kidney cells, chicken embryo fibroblast cells, and embryonated chicken eggs in a transient replication model. The results demonstrated that all three siRNAs expressing plasmids efficiently transcribed the short hairpin RNAs and inhibited expression of the NP or PA proteins measured by northern blot and western blot analyses, respectively, in the transfected cells. We also found that the integrated siRNA expression plasmid pEGFP/NP+PA, which we constructed for the first time to synchronously target NP and PA segments of the influenza virus genome, could more efficiently inhibit synthesis of influenza virus detected by cytopathogenic effects, hemagglutinin, and plaque-forming unit assays in the transfected cells. Furthermore, the integrated siRNA expression plasmid pEGFP/NP+PA could remarkably interrupt the cellular apoptotic course caused by influenza virus, which protected infected cells from apoptotic damage. In contrast, a control siRNA expression plasmid, pEGFP/HK, could neither inhibit the protein expression and production of influenza virus nor interrupt the cell apoptotic course mediated by influenza virus. These results demonstrate that RNA interference (RNAi) can be used to inhibit protein expression and replication of influenza virus and that RNAi treatment holds potential as a new approach to prevent avian influenza.

Animals↗

PubMedDate not supplied

VBP-1 is necessary for morphogenesis in Caenorhabditis elegans.

VBP-1 has been isolated as a novel protein binding to the von Hippel-Lindau (VHL) tumor suppressor gene product. The fundamental function of the gene was examined in the nematode, Caenorhabditis elegans (C. elegans). The C. elegans VBP-1 gene was expressed in adults only in the gonads and was also expressed in early stage embryos. Double-stranded RNA interference (RNAi) against VBP-1 resulted in an arrest of embryogenesis at morula stages. This suggests that the VBP-1 product is necessary for morphogenesis.

Amino Acid Sequence↗

PubMed2006

The germ line limited M element of Tetrahymena is targeted for elimination from the somatic genome by a homology-dependent mechanism.

A RNA interference (RNAi) like mechanism is involved in elimination of thousands of DNA segments from the developing somatic macronucleus of Tetrahymena, yet how specific internal eliminated sequences (IESs) are recognized remains to be fully elucidated. To define requirements for DNA rearrangement, we performed mutagenesis of the M element, a well-studied IES. While sequences within the macronucleus-retained DNA are known to determine the excision boundaries, we show that sequences internal to these boundaries are required to promote this IES's rearrangement. However, this element does not contain any specific sequence required in cis as removal of its entire left or right side was insufficient to abolish all rearrangement. Instead, rearrangement efficiency correlated with the overall size of the M element sequence within a given construct, with a lower limit of nearly 300 bp. Also, the observed minimal region necessary to epigenetically block excision supports this size limit. Truncated M element constructs that exhibited impaired rearrangement still showed full transcriptional activity, which suggests that their defect was due to inefficient recognition. This study indicates that IESs are targeted for elimination upon their recognition by homologous small RNAs and further supports the idea that DNA elimination is a RNAi-related mechanism involved in genome surveillance.

Animals↗

PubMed2005

Functional polarity is introduced by Dicer processing of short substrate RNAs.

Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes. Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3'-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3'-overhang with 3'-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3'-overhang resides on the antisense (AS) strand and dicing proceeds to the 'right' ('R') and one where the 3'-overhang resides on the sense (S) strand and dicing proceeds to the 'left' ('L'). Interestingly, the 'R' version of the asymmetric 27mer is generally more potent in reducing target gene levels than the 'L' version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the 'R' form favoring S strand and the 'L' form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.

Cell Line↗

PubMed2003

The genes orthodenticle and hunchback substitute for bicoid in the beetle Tribolium.

In Drosophila, the morphogen Bicoid organizes anterior patterning in a concentration-dependent manner by activating the transcription of target genes such as orthodenticle (otd) and hunchback (hb), and by repressing the translation of caudal. Homologues of the bicoid gene have not been isolated in any organism apart from the higher Dipterans. In fact, head and thorax formation in other insects is poorly understood. To elucidate this process in a short-germband insect, I analysed the function of the conserved genes orthodenticle-1 (otd-1) and hb in the flour beetle Tribolium castaneum. Here I show that, in contrast to Drosophila, Tribolium otd-1 messenger RNA is maternally inherited by the embryo. Reduction of Tribolium otd-1 levels by RNA interference (RNAi) results in headless embryos. This shows that otd-1 is required for anterior patterning in Tribolium. As in Drosophila, Tribolium hb specifies posterior gnathal and thoracic segments. The head, thorax and the anterior abdomen fail to develop in otd-1/hb double-RNAi embryos. This phenotype is similar to that of strong bicoid mutants in Drosophila. I propose that otd-1 and hb are part of an ancestral anterior patterning system.

Animals↗

PubMed2003

RNA interference as a metabolic engineering tool: potential for in vivo control of protein expression in an insect larval model.

Many ex vivo factors influence the yield of recombinant protein produced via AcMNPV (Autographa californica multiple nucleocapsid nuclear polyhedrosis virus) in Trichoplusia ni (T. ni) larvae. Among these are: the method of infection, the time of infection, the virus load, and the time of harvest. In vivo strategies, however, that attempt to manipulate host function in this and other expression systems have largely been ignored. In this work, RNA interference (RNAi) is shown as an effective metabolic engineering controller to downregulate targeted gene expression. Specifically, RNAi was made to virus-encoded gfp(uv) and was found to inhibit the production of GFPuv in larvae when injected within an 18-h window (before and after) of baculovirus infection. The level of inhibition was found to depend, both in duration and extent, on the concentration of injected RNAi. That relatively low levels of RNAi can inhibit protein synthesis driven by the strong polyhedrin (polh) promoter of AcMNPV, suggests that RNAi will find utility as an in vivo metabolic controller in metabolic engineering studies such as this one pertaining to protein expression.

Animals↗

PubMed2005

RNA interference: ready to silence cancer?

RNA interference (RNAi) is considered the most promising functional genomics tool recently developed. As in other medical fields, this biotechnology might revolutionize the approach to dissecting the biology of cancer, ultimately speeding up the discovery pace of novel targets suitable for molecularly tailored antitumor therapies. In addition, preclinical results suggest that RNAi itself might be used as a therapeutic weapon. With the aim of illustrating not only the potentials but also the current limitations of RNAi as a tool in the fight against cancer, here we summarize the physiology of RNAi, discuss the main technical issues of RNAi-based gene silencing, and review some of the most interesting preclinical results obtained so far with its implementation in the field of oncology.

Animals↗

PubMed2006

Introduction of silencing-inducing transgenes does not affect expression of known transcripts.

While the RNA interference (RNAi) mechanism has only been discovered a decade ago, RNAi is now often used to study gene function by sequence-specific knockdown of gene expression. However, it is still unknown whether introduction of silencing-inducing transgenes alters the transcriptome. To address this question, genome-wide transcriptional changes in silenced and non-silenced backgrounds were monitored through microarray analysis. No significant transcriptional changes were detected when compared to the non-silenced control. This result was confirmed by real-time polymerase chain reaction analysis of genes known to be involved in RNA silencing. In conclusion, introduction of silencing-inducing constructs does not affect expression of known transcripts in other genes than in those homologous to the targeted ones. Consequently, when gene function is studied by RNAi, the transcriptional changes detected will specifically be the result of knockout of the gene of interest, at least for the genes present on the array used in our study.

Arabidopsis↗

PubMed2006

Reduced MTA1 expression by RNAi inhibits in vitro invasion and migration of esophageal squamous cell carcinoma cell line.

To distinguish aggressive esophageal squamous cell carcinoma from indolent disease is the important clinical challenge. Studies have indicated that metastasis-associated gene 1(Mta1) played a role in the process of metastasis of carcinoma. The overexpression of Mta1 gene has been found in a variety of tumors. To identify the detailed roles of MTA1 protein in the carcinogenesis of esophageal squamous cell carcinoma, this study analyzed the pathological specimens on tissue microarray derived from 72 patients using immunohistochemistry. MTA1 expression increased in the nuclear with the development of esophageal squamous cell carcinoma from normal epithelial cell, dysplasia, to invasive cancer. In biological studies with human esophageal squamous cell carcinoma cell line, MTA1 plays its roles to promote cancer cell invasion, adhesion and movement. RNA interference (RNAi) against MTA1 decreased the malignant phenotypes. Gene microarray analysis revealed some metastasis-associated genes were altered by MTA1 RNAi. This study started an effective beginning to explore metastasis mechanisms and cancer gene therapy strategy targeting MTA1.

Base Sequence↗

PubMed2005

Modulation of gene expression by siRNA in hematopoietic cells.

RNA interference (RNAi) has been established as a powerful tool for identifying gene function in many biological processes and can be used for genome-wide functional genetic screens in mammalian cells. For such purposes, expression cassettes encoding RNAi triggers can be efficiently introduced into the host cell genome utilizing viral vector systems, resulting in long-term silencing of target gene expression. Transient gene silencing can also be induced by exogenous delivery of suitable RNAi triggers to target cells. However, similarly to other reverse genetic tools, there are technical challenges and limitations associated with RNAi, some of which are specific to hematopoietic cells. In this review we discuss the rational design of effective RNAi triggers, different approaches for their efficient delivery, and the value of RNAi both as a potential therapeutic strategy and as a tool for functional genomics and target validation in hematopoietic cells.

Animals↗

PubMed2006

Microplitis demolitor bracovirus inhibits phagocytosis by hemocytes from Pseudoplusia includens.

The braconid wasp Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of several noctuid moths. A key function of MdBV in parasitism is suppression of the host's cellular immune response. Prior studies in the host Pseudoplusia includens indicated that MdBV blocks encapsulation by preventing two types of hemocytes, plasmatocytes and granulocytes, from adhering to foreign targets. The other main immune response mediated by insect hemocytes is phagocytosis. The goal of this study was to determine which hemocyte types were phagocytic in P. includens and to assess whether MdBV infection affects this defense response. Using the bacterium Escherichia coli and inert polystyrene beads as targets, our results indicated that the professional phagocyte in P. includens is granulocytes. The phagocytic responses of granulocytes were very similar to those of High Five cells that prior studies have suggested are a granulocyte-like cell line. MdBV infection dose-dependently disrupted phagocytosis in both cell types by inhibiting adhesion of targets to the cell surface. The MdBV glc1.8 gene encodes a cell surface glycoprotein that had previously been implicated in disruption of adhesion and encapsulation responses by immune cells. Knockdown of glc1.8 expression by RNA interference (RNAi) during the current study rescued the ability of MdBV-infected High Five cells to phagocytize targets. Collectively, these results indicate that glc1.8 is a key virulence determinant in disruption of both adhesion and phagocytosis by insect immune cells.

Animals↗

PubMed2007

Arabidopsis thaliana GH3.9 influences primary root growth.

Auxins regulate a complex signal transduction network to direct plant development. Auxin-responsive genes fit into three major classes: the so-called auxin/indole-3-acetic acid (Aux/IAA), the GH3, and the small auxin-up RNA (SAUR) gene families. The 20-member Arabidopsis thaliana GH3 gene family has been subdivided into three groups. In vitro studies have shown that most Group II members function as IAA-amido synthetases to conjugate amino acids to the plant hormone auxin. Here we report the role of a previously uncharacterized GH3 gene family member, GH3.9, in root growth. Unlike most other Group II family members, GH3.9 expression was repressed by low concentrations of exogenous IAA in seedlings. Transgenic plants harboring a GH3.9 promoter::reporter gene construct indicate that GH3.9 is expressed in the root-hypocotyl junction, leaves and the shoot apical meristem of young seedlings, in mature embryos, and in the root vascular tissue. Expression was also observed in lateral root tips when seedlings were treated with exogenous IAA. Inverse PCR was used to identify an activation tagged T-DNA insertion in chromosome 2 near the 5'UTR region of At2g47750 (GH3.9). Plants homozygous for the T-DNA insertion (gh3.9-1 mutants) had reduced GH3.9 expression, no obvious effects on apical dominance or leaf morphology, greater primary root length, and increased sensitivity to indole-3-acetic acid (IAA)-mediated root growth inhibition. Additional T-DNA insertion alleles and transgenic plants with reduced GH3.9 transcript levels due to RNA-interference (RNAi) also showed these same phenotypes. Our results provide new information on the function of GH3.9 in roots where it is likely to control auxin activity through amino acid conjugation.

Amino Acids↗

PubMed2006

Downregulation of xIAP expression by small interfering RNA inhibits cellular viability and increases chemosensitivity to methotrexate in human hepatoma cell line HepG2.

The aim of this study was to investigate whether downregulating the expression of xIAP by RNAi (RNA interference) technology can induce the apoptosis of HepG2 cells, inhibit cellular viability and increase chemosensitivity of cancer cells. HepG2 cells were transfected with U6 promoter plasmids coding for short interfering RNAs (siRNAs) targeting xIAP. RT-PCR and western blot analysis were used to assess the mRNA and protein levels of xIAP expression. T he suppression efficiency o f xIAPby RNAi was evaluated using the MTT assay for cellular viability and Annexin V-PI binding assay for apoptosis. These results showed that siRNAs reduced cellular viability and increased cellular apoptosis. Moreover, downregulation of xIAP expression enhanced the chemosensitivity of HepG2 cells to methotrexate. These results suggest that the downregulation of xIAP by RNAi could potentially be used as a therapeutic strategy for human hepatocellular carcinoma.

Antimetabolites, Antineoplastic↗

PubMed2005

RNA interference against enterovirus 71 infection.

Enterovirus 71 (EV71) is a highly infectious major causative agent of hand, foot, and mouth disease (HFMD) which could lead to severe neurological complications. There is currently no effective therapy against EV71. In this study, RNA interference (RNAi) is employed as a therapeutic approach for specific viral inhibition. Various regions of the EV71 genome were targeted for inhibition by chemically synthesized siRNAs. Transfection of rhabdomyosarcoma (RD) cells with siRNA targeting the 3'UTR, 2C, 3C, or 3D region significantly alleviated cytopathic effects of EV71. The inhibitory effect was dosage-dependent with a corresponding decrease in viral RNA, viral proteins, and plaque formations by EV71. Viral inhibition of siRNA transfected RD cells was still evident after 48 h. In addition, no significant adverse off-target silencing effects were observed. These results demonstrated the potential and feasibility for the use of siRNA as an antiviral therapy for EV71 infections.

Base Sequence↗

PubMed2007

Effect of siRNA nuclease stability on the in vitro and in vivo kinetics of siRNA-mediated gene silencing.

Small interfering RNA (siRNA) molecules achieve sequence-specific gene silencing through the RNA interference (RNAi) mechanism. Here, live-cell and live-animal bioluminescent imaging (BLI) is used to directly compare luciferase knockdown by unmodified and nuclease-stabilized siRNAs in rapidly (HeLa) and slowly (CCD-1074Sk) dividing cells to reveal the impact of cell division and siRNA nuclease stability on the kinetics of siRNA-mediated gene silencing. Luciferase knockdown using unmodified siRNAs lasts approximately 1 week in HeLa cells and up to 1 month in CCD-1074Sk cells. There is a slight increase in the duration of luciferase knockdown by nuclease-stabilized siRNAs relative to unmodified siRNAs after cationic lipid transfection, but this difference is not observed after electroporation. In BALB/cJ mice, a fourfold increase in maximum luciferase knockdown is observed after hydrodynamic injection (HDI) of nuclease-stabilized siRNAs relative to unmodified siRNAs, yet the overall kinetics of the recovery after knockdown are nearly identical. By using a mathematical model of siRNA-mediated gene silencing, the trends observed in the experimental data can be duplicated by changing model parameters that affect the stability of the siRNAs before they reach the cytosolic compartment. Based on these findings, we hypothesize that the stabilization advantages of nuclease-stabilized siRNAs originate primarily from effects prior to and during internalization before the siRNAs can interact with the intracellular RNAi machinery.

Animals↗