Showing papers on "Small hairpin RNA published in 2006"
TL;DR: A screen based on high-content imaging was developed to identify genes required for mitotic progression in human cancer cells and applied to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes, providing a widely applicable resource for loss-of-function screens.
1,760 citations
TL;DR: The risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by the report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.
Abstract: RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.
1,683 citations
TL;DR: This work uses short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions, and focuses on transcriptional regulators and identifies seven genes for which shRNA-mediated depletion negatively affects self-Renewal.
Abstract: We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem cells. We use short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven genes for which shRNA-mediated depletion negatively affects self-renewal, including four genes with previously unrecognized roles in self-renewal. Perturbations of these gene products are combined with dynamic, global analyses of gene expression. Our studies suggest specific biological roles for these molecules and reveal the complexity of cell fate regulation in embryonic stem cells.
1,009 citations
TL;DR: In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with dependence on siRNA concentration similar to that of silencing of the target transcript.
Abstract: Transfected siRNAs and miRNAs regulate numerous transcripts that have only limited complementarity to the active strand of the RNA duplex. This process reflects natural target regulation by miRNAs, but is an unintended ("off-target") consequence of siRNA-mediated silencing. Here we demonstrate that this unintended off-target silencing is widespread, and occurs in a manner reminiscent of target silencing by miRNAs. A high proportion of unintended transcripts silenced by siRNAs showed 3' UTR sequence complementarity to the seed region of the siRNA. Base mismatches within the siRNA seed region reduced the set of original off-target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence. An inducible shRNA silenced a subset of transcripts that were silenced by an siRNA of the same sequence, demonstrating that unintended silencing is sequence mediated and is independent of delivery method. In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with dependence on siRNA concentration similar to that of silencing of the target transcript. Thus, short stretches of sequence complementarity to the siRNA or shRNA seed region are key to the silencing of unintended transcripts.
962 citations
TL;DR: A loss-of-function screen for genes required for the proliferation and survival of cancer cells using an RNA interference library is described and uncovered CARD11 as a key upstream signalling component responsible for the constitutive IκB kinase activity in activated B-cell-like DLBCL.
Abstract: The pursuit of novel therapeutic agents in cancer relies on the identification and validation of molecular targets. Hallmarks of cancer include self-sufficiency in growth signals and evasion from apoptosis; genes that regulate these processes may be optimal for therapeutic attack. Here we describe a loss-of-function screen for genes required for the proliferation and survival of cancer cells using an RNA interference library. We used a doxycycline-inducible retroviral vector for the expression of small hairpin RNAs (shRNAs) to construct a library targeting 2,500 human genes. We used retroviral pools from this library to infect cell lines representing two distinct molecular subgroups of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like DLBCL and germinal centre B-cell-like DLBCL. Each vector was engineered to contain a unique 60-base-pair 'bar code', allowing the abundance of an individual shRNA vector within a population of transduced cells to be measured using microarrays of the bar-code sequences. We observed that a subset of shRNA vectors was depleted from the transduced cells after three weeks in culture only if shRNA expression was induced. In activated B-cell-like DLBCL cells, but not germinal centre B-cell-like DLBCL cells, shRNAs targeting the NF-kappaB pathway were depleted, in keeping with the essential role of this pathway in the survival of activated B-cell-like DLBCL. This screen uncovered CARD11 as a key upstream signalling component responsible for the constitutive IkappaB kinase activity in activated B-cell-like DLBCL. The methodology that we describe can be used to establish a functional taxonomy of cancer and help reveal new classes of therapeutic targets distinct from known oncogenes.
604 citations
Patent•
04 Aug 2006TL;DR: In this article, the authors present methods and reagents useful for modulating gene expression in various applications such as therapeutic, diagnostic, target assessment and genome discovery applications, which relates to synthetic chemically modified small nucleic acid molecules capable of mediating RNA interference (RNAi) to a target NCA sequence, such as short interfering nucleic acids (siNA), short interfering RNA (siRNA).
Abstract: The present invention relates to methods and reagents useful for modulating gene expression in various applications such as therapeutic, diagnostic, target assessment and genome discovery applications. Specifically, the present invention relates to synthetic chemically modified small nucleic acid molecules capable of mediating RNA interference (RNAi) to a target nucleic acid sequence, such as short interfering nucleic acids (siNA), short interfering RNA (siRNA), double stranded RNA (dsRNA). ), MicroRNA (miRNA), and short hairpin RNA (shRNA) molecules. Small nucleic acid molecules are useful in the treatment of any disease or condition that responds to regulation of gene expression in a cell, tissue or organism.
475 citations
TL;DR: HIF-1 contributes to the epithelial-mesenchymal transition in VHL-null RCC by indirect repression of E-cadherin, a critical event in the pathogenesis of invasive and metastatic cancer.
Abstract: A critical event in the pathogenesis of invasive and metastatic cancer is E-cadherin loss of function. Renal clear cell carcinoma (RCC) is characterized by loss of function of the von Hippel-Lindau tumor suppressor (VHL), which negatively regulates hypoxia-inducible factor-1 (HIF-1). Loss of E-cadherin expression and decreased cell-cell adhesion in VHL-null RCC4 cells were corrected by enforced expression of VHL, a dominant-negative HIF-1α mutant, or a short hairpin RNA directed against HIF-1α. In human RCC biopsies, expression of E-cadherin and HIF-1α was mutually exclusive. The expression of mRNAs encoding TCF3, ZFHX1A, and ZFHX1B, which repress E-cadherin gene transcription, was increased in VHL-null RCC4 cells in a HIF-1–dependent manner. Thus, HIF-1 contributes to the epithelial-mesenchymal transition in VHL-null RCC by indirect repression of E-cadherin . (Cancer Res 2006; 66(5): 2725-31)
403 citations
TL;DR: It is shown that knockdown of Smad4 in MDA-MB-231 breast cancer cells strongly inhibited the frequency of bone metastasis in nude mice by 75% and significantly increased metastasis-free survival.
Abstract: Transforming growth factor beta (TGF-beta) can act as suppressor and promoter of cancer progression. Intracellular Smad proteins (i.e., receptor regulated Smads and common mediator Smad4) play a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-beta, but their function in TGF-beta-induced invasion and metastasis is unclear. Here, we have investigated the role of Smad4 in a cellular and mouse model for TGF-beta-induced breast cancer progression. Consistent with its tumor suppressor function, specific silencing of Smad4 in NMuMG mammary gland epithelial cells using small hairpin RNA (shRNA)-expressing RNAi vectors strongly mitigated TGF-beta-induced growth inhibition and apoptosis. Smad4 knockdown also potently inhibited TGF-beta-induced epithelial to mesenchymal transition of NMuMG cells as measured by morphologic transformation from epithelial to fibroblast-like cells, formation of stress fibers, inhibition of E-cadherin expression, and gain of expression of various mesenchymal markers. Furthermore, we show that knockdown of Smad4 in MDA-MB-231 breast cancer cells strongly inhibited the frequency of bone metastasis in nude mice by 75% and significantly increased metastasis-free survival. Communication of MDA-MB-231 cells with the bone microenvironment, which is needed for optimal tumor cell growth and metastasis, may be affected in Smad4 knockdown cells as TGF-beta-induced expression of interleukin 11 was attenuated on Smad4 knockdown. Taken together, our results show that Smad4 plays an important role in both tumor suppression and progression of breast cancer cells.
358 citations
TL;DR: The development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system is described.
Abstract: RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naive cell system In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Gα12 and Gα13 both singly and in combination, demonstrating the Gα13 dependence of serum response element-mediated transcription In RAW2647 macrophages, regulated knockdown of Gβ2 correlated with a reduced Ca2+ response to C5a Insertion of a GFP transgene upstream of the Gβ2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system
326 citations
TL;DR: New RNA polymerase II expression vectors for RNAi are developed, designated SIBR vectors, based upon the non-coding RNA BIC, and it is found that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells.
Abstract: Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem–loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications.
315 citations
TL;DR: RNAi has great potential as an antiviral gene therapy approach and support the efforts to develop this strategy for treatment of HIV-1-infected individuals, as well as identify multiple shRNAs that act as potent inhibitors of virus replication.
TL;DR: It is shown that bacteria engineered to produce a short hairpin RNA (shRNA) targeting a mammalian gene induce trans-kingdom RNAi in vitro and in vivo, and the potential of bacteria-mediated RNAi for functional genomics, therapeutic target validation and development of clinically compatible RNAi-based therapies is suggested.
Abstract: RNA-interference (RNAi) is a potent mechanism, conserved from plants to humans for specific silencing of genes, which holds promise for functional genomics and gene-targeted therapies. Here we show that bacteria engineered to produce a short hairpin RNA (shRNA) targeting a mammalian gene induce trans-kingdom RNAi in vitro and in vivo. Nonpathogenic Escherichia coli were engineered to transcribe shRNAs from a plasmid containing the invasin gene Inv and the listeriolysin O gene HlyA, which encode two bacterial factors needed for successful transfer of the shRNAs into mammalian cells. Upon oral or intravenous administration, E. coli encoding shRNA against CTNNB1 (catenin beta-1) induce significant gene silencing in the intestinal epithelium and in human colon cancer xenografts in mice. These results provide an example of trans-kingdom RNAi in higher organisms and suggest the potential of bacteria-mediated RNAi for functional genomics, therapeutic target validation and development of clinically compatible RNAi-based therapies.
TL;DR: It is shown that a single episode of RNAi in the nematode Caenorhabditis elegans can induce transcriptional silencing effects that are inherited indefinitely in the absence of the original trigger.
Abstract: Small RNA molecules participate in a variety of activities in the cell: in a process known as RNA interference (RNAi), double-stranded RNA triggers the degradation of messenger RNA that has a matching sequence; the small RNA intermediates of this process can also modify gene expression in the nucleus Here we show that a single episode of RNAi in the nematode Caenorhabditis elegans can induce transcriptional silencing effects that are inherited indefinitely in the absence of the original trigger Our findings may prove useful in the ongoing development of RNAi to treat disease
TL;DR: Comparison of first- and second-generation libraries indicates that RNAi triggers that enter the RNAi pathway through a more natural route yield more effective silencing.
Abstract: Loss-of-function genetics has proven essential for interrogating the functions of genes and for probing their roles within the complex circuitry of biological pathways. In many systems, technologies allowing the use of such approaches were lacking before the discovery of RNA interference (RNAi). We have constructed first-generation short hairpin RNA (shRNA) libraries modeled after precursor microRNAs (miRNAs) and second-generation libraries modeled after primary miRNA transcripts (the Hannon-Elledge libraries). These libraries were arrayed, sequence-verified, and cover a substantial portion of all known and predicted genes in the human and mouse genomes. Comparison of first- and second-generation libraries indicates that RNAi triggers that enter the RNAi pathway through a more natural route yield more effective silencing. These large-scale resources are functionally versatile, as they can be used in transient and stable studies, and for constitutive or inducible silencing. Library cassettes can be easily shuttled into vectors that contain different promoters and/or that provide different modes of viral delivery.
TL;DR: A review of siRNA delivery method strategies and the potential of RNAi‐based gene therapy in cancer treatment and the development of safe and efficacious delivery systems are presented.
Abstract: One of the most dramatic events of the past 5 years in the field of molecular biology has been the discovery of RNA interference (RNAi). Although RNAi is an evolutionarily conserved phenomenon for sequence-specific gene silencing in mammalian cells, exogenous small interfering RNA (siRNA) and vector-based short hairpin RNA (shRNA) can also invoke RNAi responses. Both are now not only experimental tools for analyzing gene function but are expected to be excellent avenues for drug target discovery and the emerging class of gene medicine for targeting incurable diseases such as cancer. The success of cancer therapeutic use of RNAi relies on the development of safe and efficacious delivery systems that introduce siRNA and shRNA expression vectors into target tumor cells. For their delivery, a variety of strategies have been used, most of them based on traditional gene therapy delivery systems. In this review, we present siRNA delivery method strategies and discuss the potential of RNAi-based gene therapy in cancer treatment.
TL;DR: The identification of a specific PP2A regulatory subunit, B56α, that selectively associates with the N terminus of c-Myc is reported, revealing a critical interconnection between a potent oncoprotein, c- myc, and a well-documented tumor suppressor,PP2A.
Abstract: Protein phosphatase 2A (PP2A) plays a prominent role in controlling accumulation of the proto-oncoprotein c-Myc. PP2A mediates its effects on c-Myc by dephosphorylating a conserved residue that normally stabilizes c-Myc, and in this way, PP2A enhances c-Myc ubiquitin-mediated degradation. Stringent regulation of c-Myc levels is essential for normal cell function, as c-Myc overexpression can lead to cell transformation. Conversely, PP2A has tumor suppressor activity. Uncovering relevant PP2A holoenzymes for a particular target has been limited by the fact that cellular PP2A represents a large heterogeneous population of trimeric holoenzymes, composed of a conserved catalytic subunit and a structural subunit along with a variable regulatory subunit which directs the holoenzyme to a specific target. We now report the identification of a specific PP2A regulatory subunit, B56alpha, that selectively associates with the N terminus of c-Myc. B56alpha directs intact PP2A holoenzymes to c-Myc, resulting in a dramatic reduction in c-Myc levels. Inhibition of PP2A-B56alpha holoenzymes, using small hairpin RNA to knock down B56alpha, results in c-Myc overexpression, elevated levels of c-Myc serine 62 phosphorylation, and increased c-Myc function. These results uncover a new protein involved in regulating c-Myc expression and reveal a critical interconnection between a potent oncoprotein, c-Myc, and a well-documented tumor suppressor, PP2A.
TL;DR: Cells suppressed for hVDAC1 but expressing either native mVDac1 or an E72Q mutant underwent apoptosis induced by various stimuli that can be inhibited by ruthenium red in the native cells but not in the mutated cells, suggesting that VDAC1 regulates apoptosis independent of the apoptosis-inducing pathway.
Abstract: Mitochondria not only generate cellular energy, but also act as the point for cellular decisions leading to apoptosis. The voltage-dependent anion channel (VDAC), as a major mitochondrial outer-membrane transporter, has an important role in energy production by controlling metabolite traffic and is also recognized as a key protein in mitochondria-mediated apoptosis. In this study, the role of VDAC1 in regulating cell survival and death was investigated by silencing endogenous human (h)VDAC1 expression by using a short hairpin RNA (shRNA)-expressing vector. The shRNA effectively down-regulated the expression in human T-REx-293 cells of hVDAC1 but not murine (m)VDAC1. Cells in which hVDAC1 expression was decreased by ≈90% proliferated extremely slowly. Normal growth was, however, restored upon expression of mVDAC1 in a tetracycline-regulated manner. Although low tetracycline concentrations promoted cell growth, high concentrations induced mVDAC1 overexpression, leading to cell death. Cells with low levels of VDAC1 showed 4-fold-lower ATP-synthesis capacity and contained low ATP and ADP levels, with a strong correlation between ATP levels and cell growth, suggesting limited metabolite exchange between mitochondria and cytosol. The possibility of suppressing endogenous hVDAC1 expression and introducing native and mutated mVDAC1 is used to further explore the involvement of VDAC1 in apoptosis. Cells suppressed for hVDAC1 but expressing either native mVDAC1 or an E72Q mutant underwent apoptosis induced by various stimuli that can be inhibited by ruthenium red in the native cells but not in the mutated cells, suggesting that VDAC1 regulates apoptosis independent of the apoptosis-inducing pathway.
TL;DR: The data validate LEDGF/p75 as an important cellular cofactor for HIV integration and as a potential target for antiviral drug development.
Abstract: After identifying the interaction between the transcriptional coactivator lens epithelium-derived growth factor (LEDGF/p75) and the human immunodeficiency virus type 1 (HIV-1) integrase (IN), we have now investigated the role of LEDGF/p75 during HIV replication. Transient small interfering RNA-mediated knockdown of LEDGF/p75 in HeLaP4 cells resulted in a three- to fivefold inhibition of HIV-1 (strain NL4.3) replication. Quantitative PCR was used to pinpoint the replication block to the integration step. Next, polyclonal and monoclonal HeLaP4-derived cell lines were selected with a stable knockdown of LEDGF/p75 mediated by a lentiviral vector (lentivector) encoding a short hairpin RNA (shRNA) targeting this protein. Cell lines stably transduced with a lentivector encoding an unrelated hairpin or a double-mismatch hairpin served as controls. Again, a two- to fourfold reduction of HIV-1 replication was observed. The extent of LEDGF/p75 knockdown closely correlated with the reduction of HIV-1 replication. After the back-complementation of LEDGF/p75 in the poly- and monoclonal knockdown cell lines using an shRNA-resistant expression plasmid, viral replication was restored to nearly wild-type levels. The Q168A mutation in integrase has been shown to interfere with the interaction with LEDGF/p75 without reducing the enzymatic activity. Transduction by HIV-1-derived lentivectors carrying the Q168A IN mutant was severely hampered, pointing again to a requirement for LEDGF/p75. Altogether, our data validate LEDGF/p75 as an important cellular cofactor for HIV integration and as a potential target for antiviral drug development.
TL;DR: The history of RNAi as well as recent discoveries are explored, highlighting recent discoveries in an intricate network of proteins that ensures the degradation of the target mRNA.
Abstract: The use of the RNA interference (RNAi) pathway to eliminate gene products has greatly facilitated the understanding of gene function. Behind this remarkable pathway is an intricate network of proteins that ensures the degradation of the target mRNA. In this review, we explore the history of RNAi as well as highlighting recent discoveries.
Patent•
14 Feb 2006
TL;DR: In this paper, formulated molecular compositions (FMC) or lipid nanoparticles (LNP) are defined for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of gene expression and/or activity in a subject or organism.
Abstract: The present invention relates to novel cationic lipids, transfection agents, microparticles, nanoparticles, and short interfering nucleic acid (siNA) molecules. The invention also features compositions, and methods of use for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of gene expression and/or activity in a subject or organism. Specifically, the invention relates to novel cationic lipids, microparticles, nanoparticles and transfection agents that effectively transfect or deliver biologically active molecules, such as antibodies (e.g., monoclonal, chimeric, humanized etc.), cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, dsRNA, allozymes, aptamers, decoys and analogs thereof, and small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, to relevant cells and/or tissues, such as in a subject or organism. Such novel cationic lipids, microparticles, nanoparticles and transfection agents are useful, for example, in providing compositions to prevent, inhibit, or treat diseases, conditions, or traits in a cell, subject or organism. The compositions described herein are generally referred to as formulated molecular compositions (FMC) or lipid nanoparticles (LNP).
TL;DR: It is shown that JNK1 is activated in response to collagen I, which increases tumorigenesis by up-regulating N-cadherin expression and by increasing motility, and that inhibiting c-Jun NH(2)-terminal kinase (JNK) with chemical inhibitors or short hairpin RNA abrogated all collagen I-induced changes.
Abstract: We have previously shown that N-cadherin expression is associated with tumor invasion, and that some cancer cells respond to specific extracellular matrix molecules by up-regulating N-cadherin. Pancreatic cancer is characterized by excessive deposition of type I collagen. Here, we show that human pancreatic cancer cells respond to collagen I, but not other matrices, by increasing motility and up-regulating mesenchymal markers, including N-cadherin. Both collagen I-mediated motility and metastasis in a mouse model for pancreatic cancer were inhibited by N-cadherin knockdown. Furthermore, inhibiting c-Jun NH(2)-terminal kinase (JNK) with chemical inhibitors or short hairpin RNA abrogated all collagen I-induced changes. We show that JNK1 is activated in response to collagen I, which increases tumorigenesis by up-regulating N-cadherin expression and by increasing motility.
TL;DR: Results provide evidence that adeno-associated virus-mediated long-term knockdown of genes can be used to delineate their effects on complex behaviors in discrete brain regions and abolish female proceptive and receptive sexual behaviors while enhancing rejection behavior.
Abstract: Estrogen receptor α (ERα) plays a major role in the regulation of neuroendocrine functions and behaviors by estrogens. Although the generation of ERα knockout mice advanced our knowledge of ERα functions, gene deletion using this method is global and potentially confounded by developmental consequences. To achieve a site-specific knockdown of ERα in the normally developed adult brain, we have generated an adeno-associated virus vector expressing a small hairpin RNA targeting ERα. After bilateral injection of this vector into the hypothalamic ventromedial nucleus in ovariectomized female mice, expression levels of ERα as well as the estrogen-inducible progesterone receptor were profoundly reduced despite the continued presence of this receptor elsewhere in the brain. Functionally, silencing of ERα in the ventromedial nucleus abolished female proceptive and receptive sexual behaviors while enhancing rejection behavior. These results provide evidence that adeno-associated virus-mediated long-term knockdown of genes can be used to delineate their effects on complex behaviors in discrete brain regions.
TL;DR: In this review, the latest developments of in vivo delivery of siRNA and the crucial issues related to this effort are addressed.
TL;DR: Together, the emerging mechanism is that EGCG blocks Wnt signaling by inducing the HBP1 transcriptional repressor and inhibits aspects of invasive breast cancer.
TL;DR: The results suggest that nutlin-3's tumor specificity may result from its ability to turn a cancer cell–specific property (activated DNA damage signaling3) into a weakness that can be exploited therapeutically.
Abstract: The identification of the cellular targets of small molecules with anticancer activity is crucial to their further development as drug candidates. Here, we present the application of a large-scale RNA interference-based short hairpin RNA (shRNA) barcode screen to gain insight in the mechanism of action of nutlin-3 (1). Nutlin-3 is a small-molecule inhibitor of MDM2, which can activate the p53 pathway. Nutlin-3 shows strong antitumor effects in mice, with surprisingly few side effects on normal tissues. Aside from p53, we here identify 53BP1 as a critical mediator of nutlin-3-induced cytotoxicity. 53BP1 is part of a signaling network induced by DNA damage that is frequently activated in cancer but not in healthy tissues. Our results suggest that nutlin-3's tumor specificity may result from its ability to turn a cancer cell-specific property (activated DNA damage signaling) into a weakness that can be exploited therapeutically.
TL;DR: This work analyzes the contributions of a novel complex member, SEL1L, mammalian homologue of yHrd3p, to the dislocation process, and implies the existence of multiple independent modes of extraction of misfolded substrates from the mammalian ER.
Abstract: Protein quality control in the endoplasmic reticulum (ER) involves recognition of misfolded proteins and dislocation from the ER lumen into the cytosol, followed by proteasomal degradation Viruses have co-opted this pathway to destroy proteins that are crucial for host defense Examination of dislocation of class I major histocompatibility complex (MHC) heavy chains (HCs) catalyzed by the human cytomegalovirus (HCMV) immunoevasin US11 uncovered a conserved complex of the mammalian dislocation machinery We analyze the contributions of a novel complex member, SEL1L, mammalian homologue of yHrd3p, to the dislocation process Perturbation of SEL1L function discriminates between the dislocation pathways used by US11 and US2, which is a second HCMV protein that catalyzes dislocation of class I MHC HCs Furthermore, reduction of the level of SEL1L by small hairpin RNA (shRNA) inhibits the degradation of a misfolded ribophorin fragment (RI332) independently of the presence of viral accessories These results allow us to place SEL1L in the broader context of glycoprotein degradation, and imply the existence of multiple independent modes of extraction of misfolded substrates from the mammalian ER
TL;DR: Modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, making primer extension the most efficient and cost-effective approach tested and inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression.
Abstract: Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20 – 40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process.
TL;DR: It is demonstrated here that lymphoid enhancer-binding factor 1 (LEF-1) mediates the proliferation, survival and differentiation of granulocyte progenitor cells and is an instructive factor regulating neutrophilic granulopoiesis whose absence plays a critical role in the defective maturation program of myeloid progenitors in individuals with CN.
Abstract: We demonstrate here that lymphoid enhancer-binding factor 1 (LEF-1) mediates the proliferation, survival and differentiation of granulocyte progenitor cells. We initially documented the importance of this transcription factor in the bone marrow of individuals with severe congenital neutropenia (CN) with a 'differentiation block' at the promyelocytic stage of myelopoiesis. LEF-1 expression was greatly reduced or even absent in CN arrested promyelocytes, resulting in defective expression of the LEF-1 target genes CCND1, MYC and BIRC5, encoding cyclin D1 (ref. 2), c-Myc and survivin, respectively. In contrast, healthy individuals showed highest LEF-1 expression in promyelocytes. Reconstitution of LEF-1 in early hematopoietic progenitors of two individuals with CN corrected the defective myelopoiesis and resulted in the differentiation of these progenitors into mature granulocytes. Repression of endogenous LEF-1 by specific short hairpin RNA inhibited proliferation and induced apoptosis of CD34(+) progenitors from healthy individuals and of cells from two myeloid lines (HL-60 and K562). C/EBPalpha, a key transcription factor in granulopoiesis, was directly regulated by LEF-1. These observations indicate that LEF-1 is an instructive factor regulating neutrophilic granulopoiesis whose absence plays a critical role in the defective maturation program of myeloid progenitors in individuals with CN.
TL;DR: Small interfering RNA screens were used to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics and found tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.
Abstract: Gene silencing by RNA interference is a powerful genetic tool to identify genes involved in specific biological processes in model organisms and human cells (3, 14, 18, 38, 42, 56). It is now possible to perform unbiased genetic screens using cultured human cells. Recently, small-scale siRNA (small interfering RNA) and shRNA (small hairpin RNA) screens in human cells have successfully identified genes that modulate cell growth, apoptosis, chemoresistance, and chemosensitivity (4, 44, 52). While establishing proof-of-concept, most of these early screens tested gene sets too limited in size to provide a comprehensive view of the genes involved in the cellular phenotypes being tested. Several groups have employed nearly genome-wide screens of shRNAs expressed from vectors to identify genes that modify certain phenotypes (6, 40, 49, 59). To date, these shRNA screens have been limited to phenotypes amenable to positive selection, but other approaches like bar code screening have shown promise (6). Systematic well-by-well testing of genomic-scale siRNA or shRNA libraries for modulation of nonselectable phenotypes in human cells has not been widely attempted.
Cancer cells undergo numerous genetic changes that drive cellular transformation from normal cell progenitors. Anticancer drug target discovery is now frequently directed toward understanding and exploiting genetic alterations that exist in tumor cells. Knowledge of genetic alterations in tumors may lead to better use of conventional therapeutics or development of new therapeutics that offer better therapeutic windows. Loss-of-function genetic screens can identify genes whose loss of function enhances cytotoxicity of chemotherapeutics in cells with defined genetic lesions.
The TP53 transcription factor is a central mediator of the cellular response to DNA damage and a variety of other stresses and is one of the most frequently mutated genes in human cancers (19, 57). TP53 status can also effect the chemosensitivity of tumor cells (15). The identification of genes that, when silenced, selectively enhance the chemosensitivity of TP53 mutant cancer cells but not TP53 wild-type cells would make attractive drug targets. Drugs developed to these genes have the potential to selectively increase the toxicity of the chemotherapeutic in the cancer cell.
Genetic screens in model organisms have led to identification of drug enhancers whose loss increases sensitivity to the drug (43, 54, 56, 61). We hypothesized that identification of enhancers for commonly used cancer therapeutics would lead to new strategies for therapeutic application of these drugs and/or identification of new targets for the development of combination chemotherapies. Here we used small- and genome-scale siRNA screens to identify genes that enhance the sensitivity of human tumor cells to the cancer chemotherapeutic cisplatin. The screen hits included known genes that function in DNA repair, cell cycle checkpoints, and survival signaling, as well as genes with no annotated functions. Selected hits from these screens were validated and then tested on matched TP53-positive and TP53-deficient cells to identify enhancers selective for tumor cells that have lost TP53 function. We show that silencing of BRCA1, BRCA2, and certain genes whose products interact with BRCA1/2, selectively enhances cisplatin cytotoxicity in TP53-deficient cells.
TL;DR: It is shown that several proteins previously recognized to play a role in a specific RNA stem-loop function in cis were also linked to RNA silencing pathways where a different type of hairpin acts in trans, indicating that the relationship between certain mechanisms that recognize different types of RNA hairpins is closer than previously thought.
Abstract: An RNA hairpin is an essential secondary structure of RNA. It can guide RNA folding, determine interactions in a ribozyme, protect messenger RNA (mRNA) from degradation, serve as a recognition motif for RNA binding proteins or act as a substrate for enzymatic reactions. In this review, we have focused on cis-acting RNA hairpins in metazoa, which regulate histone gene expression, mRNA localization and translation. We also review evolution, mechanism of action and experimental use of trans-acting microRNAs, which are coded by short RNA hairpins. Finally, we discuss the existence and effects of long RNA hairpin in animals. We show that several proteins previously recognized to play a role in a specific RNA stem-loop function in cis were also linked to RNA silencing pathways where a different type of hairpin acts in trans. Such overlaps indicate that the relationship between certain mechanisms that recognize different types of RNA hairpins is closer than previously thought.