Search

Aims: Mice with a cardiomyocyte (CM)-restricted knockout of signal transducer and activator of transcription 3 (STAT3-KO) develop spontaneous heart failure. We investigated the impact of STAT3-mediated regulation of microRNAs for pathophysiological alterations in the heart., Methods and Results: MicroRNAchip and qRT-PCR analysis revealed elevated cardiac expression of miR-199a in STAT3-KO mice. Lentiviral shRNA-mediated STAT3-knock-down in neonatal rat CMs markedly increased miR-199a promoter activity and miR-199a levels indicative of a suppressive effect of STAT3 on miR-199a transcription. Up-regulated miR-199a in CM by pre-miR-199a transfection (pre-miR-199a-CM) reduced expression of components of the ubiquitin-proteasome system (UPS), i.e. the ubiquitin-conjugating enzymes Ube2g1 (mRNA and protein) and Ube2i (protein). Pre-miR-199a-CM or CM with siRNA-mediated down-regulation of Ube2i and Ube2g1 (siRNA-Ube2i/2g1-CM) displayed massive down-regulation of α- and β-myosin heavy chain expression associated with disrupted sarcomere structures. In addition, protein arginine methyltransferase I (PRMT-I) expression and asymmetric dimethylarginine (ADMA) synthesis were increased in pre-miR-199a-CM or in siRNA-Ube2i/2g1-CM. Increased ADMA in cell culture supernatant (SN) from pre-miR-199a-CM or siRNA-Ube2i/2g1-CM lowered nitric oxide (NO) bioavailability of rat cardiac endothelial cells while lowering ADMA concentration in CM SNs by the PRMT inhibitor arginine methyltransferase inhibitor 1 (AMI-1) (100 µM) improved NO bioavailability. In STAT3-KO hearts Ube2i and Ube2g1 expression were markedly reduced. Human terminal failing hearts harbouring low STAT3 protein levels displayed increased miR-199a levels and decreased Ube2g1 expression., Conclusion: This study identifies a novel pathophysiological circuit in the heart between reduced STAT3 protein levels, increased miR-199a expression, and subsequent impairment of the UPS that disrupts CM sarcomere structure and impairs via the release of ADMA endothelial cell function.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-, maturation-, and disease-specific miRNA expression has been described, miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics, biochemical analysis, and unbiased and hypothesis-driven miRNA target prediction, we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells, miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression, respectively. However, gene-specific RNAi reveals that this reduction, alone and in combination, is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression, DNA-binding, and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression, indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed, we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly, gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells.

Micro (mi)RNAs are highly conserved small regulatory RNAs, which regulate gene expression by hybridization to specific binding sites in the 3'untranslated region (UTR) of many mRNAs. Upon miRNA-guided recruitment of a multiprotein complex, target mRNAs are either degraded or their translation is blocked depending on the complementarity between the miRNAs and their binding sites in target mRNAs. Individual miRNAs have been shown to regulate the expression of hundreds of genes with corresponding miRNA binding sites in the 3'UTR in a dose-dependent manner. Although miRNA-target genes may be predicted by bioinformatic tools, each potential target needs to be confirmed experimentally. We describe here the expression of individual miRNAs or miRNA-specific antagomiRs by lentiviral gene transfer to induce stable gain- and loss-of-function phenotypes. These techniques provide some tools to analyze miRNA function in cell culture or animal models.

MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation via hybridisation to mRNAs. miRNA function can be inhibited by the so-called "antagomirs" - anti-sense RNA oligonucleotides complementary to individual miRNAs. Since, in principle, any miRNA can be silenced, antagomirs provide a powerful tool to investigate the function of particular miRNAs. However, conventional methods to deliver antagomirs into cells (e.g. transfection) have been shown to only transiently interfere with endogenous miRNA expression and/or function. In this section, we describe a lentivirus-based system for stable antagomir expression to generate longterm loss-of-function phenotypes for individual miRNAs. Moreover, the described strategy is also suitable for studying the function of individual miRNAs encoded within polycistronic clusters.This chapter provides a collection of protocols to antagonize miRNA function by lentiviral antagomir expression, which can be achieved in a wide range of target cells.

RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) that results either in degradation of homologues mRNAs or inhibition of mRNA translation. The effector molecules which activate the RNAi pathway are small regulatory RNAs including small interfering RNAs (siRNAs) which are processed from longer dsRNAs by the RNAse III enzyme Dicer, and microRNAs (miRNAs) generated in a regulated multistep process from endogenous primary transcripts (pri-miRNA). Since, in principle, any gene can be silenced, RNAi provides a powerful tool to investigate gene function, and it is therefore a widely used gene silencing method in functional genomics. This chapter provides a collection of protocols for specific gene knock-down in hematopoietic cells by the application of short-hairpin RNAs (shRNAs) transcribed by RNA polymerase III (pol III) promoters or artificial-miRNAs (art-miRNAs) expressed from RNA pol II promoters using lentiviral vectors, respectively.

Micro RNAs (miRNA) regulate gene expression by hybridization and recruitment of multi-protein complexes to complementary mRNA target sequences. miRNA function can transiently be antagonized by antagomirs-chemically modified oligonucleotides complementary to individual miRNAs. Here, we describe the induction of stable loss-of-function phenotypes for specific miRNAs by lentivirus-mediated antagomir expression. Lentivirally expressed antagomirs are transcribed from a H1-promoter located within the lentiviral 3'LTR and were directed against miRNAs encoded on the polycistronic miR17-92 transcript. Functional silencing of miR-18a, miR-19b and miR-20a by the corresponding antagomirs specifically relieves miRNA-mediated reporter gene repression. Inhibition of miRNA function correlates to reduction of 'miRNA' amplification by miRNA-specific quantitative RT-PCR. Furthermore, protein expression of E2F-1, a known miR-20 target, is enhanced by lentivirally expressed anti-miR-20 antagomirs in a dose-dependent manner, whereas over-expression of miR-20a reduces E2F-1 levels. Finally, combined over-expression of specific miRNAs and antagomirs reveals individual and complementary functions of miR-18a and miR-20a and demonstrates specific miRNA impact on cell proliferation in a cell culture model.

Although targeting the BCR-ABL tyrosine kinase activity by imatinib mesylate has rapidly become first-line therapy in chronic myeloid leukemia (CML), drug resistance suggests that combination therapy directed to a complementing target may significantly improve treatment results. To identify such potential targets, we used lentivirus-mediated RNA interference (RNAi) as a tool for functional genomics in cell lines as well as primary normal and CML CD34+ cells. In a conditional cell culture model, we demonstrate that RNAi-mediated reduction of SHP2, STAT5, and Gab2 protein expression inhibits BCR-ABL-dependent but not cytokine-dependent proliferation in a dose-dependent manner. Similarly, colony formation of purified primary CML but not of normal CD34+ colony-forming cells is specifically reduced by inhibition of SHP2, STAT5, and Gab2 expression, respectively. In addition, coexpression of both anti-BCR-ABL and anti-SHP2 shRNAs from a single lentiviral vector induces stronger inhibition of colony formation as compared to either shRNA alone. The data indicate that BCR-ABL expression may affect the function of normal signaling molecules. Targeting these molecules may harbor significant therapeutic potential for the treatment of patients with CML.

In the past few years, the discovery of RNA-mediated gene silencing mechanisms, like RNA interference (RNAi), has revolutionized our understanding of eukaryotic gene expression. These mechanisms are activated by double-stranded RNA (dsRNA) and mediate gene silencing either by inducing the sequence-specific degradation of complementary mRNA or by inhibiting mRNA translation. RNAi now provides a powerful experimental tool to elucidate gene function in vitro and in vivo, thereby opening new exciting perspectives in the fields of molecular analysis and eventually therapy of several diseases such as infections and cancer. In hematology, numerous studies have described the successful application of RNAi to better define the role of oncogenic fusion proteins in leukemogenesis and to explore therapeutic approaches in hematological malignancies. In this review, we highlight recent advances and caveats relating to the application of this powerful new methodology to hematopoiesis.

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.

Mutation or dysregulation of related homeobox genes occurs in leukemia. Using RT-PCR, we screened members of the EHG family of homeobox genes, comprising EN1 (at 2q14), GBX2 (at 2q36), and EN2, GBX1, and HLXB9 (at 7q36), for dysregulation in acute myeloid leukemia (AML) cell lines indicated by chromosomal breakpoints at these sites. Only one EHG-family gene was expressed, HLXB9, in cell line GDM-1 (AML-M4). Karyotypic analysis of GDM-1 revealed a unique t(6;7)(q23;q35), also present in the patient. Fluorescence in situ hybridization analysis showed chromosomal breakpoints close to the region upstream of HLXB9, at 7q36, a region rearranged in certain AML patients, and at 6q23 upstream of MYB, a gene activated in leukemia. Detailed expression analysis suggested ectopic activation of HLXB9 occurred via juxtaposition with regions upstream of MYB, which was highly expressed in GDM-1. Our data identified a cell line model for a novel leukemic translocation involving MYB with HLXB9, further implicating HLXB9 in leukemogenesis., ((c) 2004 Wiley-Liss, Inc.)

There has been a lack of powerful tools for systematic analysis of mammalian gene function, but RNA interference (RNAi) may now provide such a strategy. Stable transcription of RNAi triggers from suitable expression cassettes integrated into the host cell genome by viral gene transfer can induce long-term and heritable gene silencing in mammalian cells. However, the use of RNAi as a genetic tool is limited by difficulties in identifying efficient RNAi triggers, the problem of effective delivery and off-target effects, as well as potential genotoxic side effects of viral gene transfer strategies. Recent insights into the molecular mechanisms of silencing processes mediated by either siRNA or miRNA will allow further optimization of RNAi triggers as genetic tools.

Small interfering RNAs (siRNAs) were designed to target the bcr-abl oncogene, which causes chronic myeloid leukemia (CML) and bcr-abl-positive acute lymphoblastic leukemia (ALL). Chemically synthesized anti-bcr-abl siRNAs were selected using reporter gene constructs and were found to reduce bcr-abl mRNA up to 87% in bcr-abl-positive cell lines and in primary cells from CML patients. This mRNA reduction was specific for bcr-abl because c-abl and c-bcr mRNA levels remained unaffected. Furthermore, protein expression of BCR-ABL and of laminA/C was reduced by specific siRNAs up to 80% in bcr-abl-positive and normal CD34(+) cells, respectively. Finally, anti-bcr-abl siRNA inhibited BCR-ABL-dependent, but not cytokine-dependent, proliferation in a bcr-abl-positive cell line. These data demonstrate that siRNA can specifically and efficiently interfere with the expression of an oncogenic fusion gene in hematopoietic cells.

Prolonged exposure of human hematopoietic stem cells (HSC) to growth factors for efficient transduction by murine oncoretroviral vectors has major detrimental effects on repopulating activity. In this study, we have used a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped human immunodeficiency virus type 1 (HIV-1) lentiviral-based vector system to transduce cord blood (CB) CD34+ cells over a limited time period (< or =24 hours). Under these conditions, significant gene marking was observed in engrafted human lymphoid, myeloid, and progenitor cells in all transplanted Severe Combined Immunodeficient (SCID) mice. To enhance the level of gene expression in hematopoietic cells, we also generated a series of lentiviral vectors incorporating the spleen focus forming virus (SFFV) long terminal repeat (LTR) sequences, and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). By including the central polypurine tract (cPPT) sequence of HIV-1 we were then able to achieve high levels of transduction (over 80%) and gene expression in vivo after a single exposure to viral supernatant. These results demonstrate that lentiviral vectors are highly effective for gene transfer to human HSC, and that SFFV regulatory sequences can be successfully incorporated to enhance the long-term expression of a transgene in primary human hematopoietic cells in vivo.

The central nervous system is a very attractive target for new therapeutic strategies since many genes involved in neurological diseases are known and often only local low level gene expression is required. However, as the blood brain barrier on one hand prevents some therapeutic agents given systematically from exerting their activity in the CNS, it also provides an immune privileged environment. Neurosurgical technology meanwhile allows the access of nearly every single centre of the CNS and provides the surgical tool for direct gene delivery via minimal invasive surgical approaches to the brain. Successful therapy of the central nervous system requires new tools for delivery of therapeutics in vitro and in vivo (Fig. 1). The application of therapeutic proteins via pumps into the CSF was shown to be only of limited value since the protein mostly is not sufficiently transported within the tissue and the half life of proteins limits the therapeutic success. Direct gene delivery into the host cell has been a main strategy for years, and in the beginning the direct DNA delivery or encapsulation in liposomes or other artificial encapsulation have been applied with different success. For several years the most promising tools have been vectors based on viruses. Viruses are able to use the host cell machinery for protein synthesis, and some of them are able to stably insert into the host cell genome and provide long term transgene expression as long as the cell is alive. The increasing knowledge of viruses and their live cycle promoted the development of viral vectors that function like a shuttle to the cell, with a single round of infection either integrating or transiently expressing the transgene. Viral vectors have proven to be one of the most efficient and stable transgene shuttle into the cell and have gained increasing importance. The limitations of some viral vectors like the adenoviral vector and adeno-associated viral vector have been improved by new constructs like HIV-1 based lentiviral vectors. The immune response caused by expression of viral proteins, or the inability of some viral vectors like the retroviral vector to infect only dividing cells have been overcome by these new constructs. Lentiviral vectors allow an efficient and stable transgene expression over years in vivo without effecting transgene expression or immune response. In this Chapter we will describe synthetic vectors, give an overview of the most common viral vectors and focus our attention on lentiviral vectors, since we consider them to be the most efficient tool for gene delivery in the CNS.