ABSTRACT
microRNA-155 (miR-155) has been implicated as a central regulator of the immune system, but its function during acute inflammatory responses is still poorly understood. Here we show that exposure of cultured macrophages and mice to lipopolysaccharide (LPS) leads to up-regulation of miR-155 and that the transcription factor c/ebp Beta is a direct target of miR-155. Interestingly, expression profiling of LPS-stimulated macrophages combined with overexpression and silencing of miR-155 in murine macrophages and human monocytic cells uncovered marked changes in the expression of granulocyte colony-stimulating factor (G-CSF), a central regulator of granulopoiesis during inflammatory responses. Consistent with these data, we show that silencing of miR-155 in LPS-treated mice by systemically administered LNA-antimiR results in derepression of the c/ebp Beta isoforms and down-regulation of G-CSF expression in mouse splenocytes. Finally, we report for the first time on miR-155 silencing in vivo in a mouse inflammation model, which underscores the potential of miR-155 antagonists in the development of novel therapeutics for treatment of chronic inflammatory diseases.
Subject(s)
CCAAT-Enhancer-Binding Protein-beta/genetics , Gene Expression Regulation , Granulocyte Colony-Stimulating Factor/genetics , Inflammation/genetics , MicroRNAs/physiology , Animals , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cell Line , Down-Regulation , Female , Gene Silencing , Granulocyte Colony-Stimulating Factor/metabolism , Humans , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/immunology , Mice , Mice, Inbred C57BL , MicroRNAs/antagonists & inhibitors , MicroRNAs/biosynthesis , Protein Biosynthesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Spleen/immunologyABSTRACT
The ability to regulate the cellular homeostasis of a higher organism through tight control of apoptosis and cell division is crucial for life. Dysregulation of these mechanisms is often associated with cancerous phenotypes in cells. Optimal cancer therapy is a fine balance between effective cancer cell killing and at the same time minimizing, or avoiding, damage to the surrounding healthy tissue. To obtain this, it is necessary to identify and inhibit molecular targets on which the cancer cells are strongly dependent. Survivin represents such a target, and it has been published previously that peptide vaccines, the small-molecule YM155, and the antisense molecule LY2181308/ISIS23722, via different mechanisms, have been used as survivin inhibitors. In this article, a new potent antisense inhibitor of survivin, SPC3042, is presented, and the properties of SPC3042 are compared with the previously published antisense drug, LY2181308/ISIS23722. SPC3042 is a 16-mer locked nucleic acid (LNA) oligonucleotide and designed as a fully phosphorothiolated gapmer containing 7 LNA nucleotides in the flanks. The LNA nucleotides in SPC3042 provide nuclease stability and higher potency for survivin mRNA inhibition compared with earlier generations of antisense reagents. It is shown that the down-regulation of survivin with SPC3042 leads to cell cycle arrest, pronounced cellular apoptosis, and down-regulation of Bcl-2. It is also shown that SPC3042 is a sensitizer of prostate cancer cells to Taxol treatment in vitro and in vivo.
Subject(s)
Apoptosis Regulatory Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Oligodeoxyribonucleotides/pharmacology , Animals , Apoptosis/drug effects , Base Sequence , Caspases/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Deoxyribonucleases/metabolism , Flow Cytometry , Gene Expression Regulation, Neoplastic/drug effects , Humans , Inhibitor of Apoptosis Proteins , Male , Mice , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Molecular Sequence Data , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Nucleic Acid Denaturation/drug effects , Paclitaxel/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Survivin , Temperature , Xenograft Model Antitumor AssaysABSTRACT
The synthesis of LNA phosphoramidates is presented. The LNA phosphoramidates were evaluated for their ability to inhibit cell proliferation of the human prostate cancer cell line 15PC3. A number of the LNA phosphoramidates showed cell proliferation inhibition determined by the MTS assay.
Subject(s)
Amides/chemical synthesis , Amides/pharmacology , Cell Proliferation/drug effects , Nucleotides/chemistry , Phosphoric Acids/chemical synthesis , Phosphoric Acids/pharmacology , Ribose/analogs & derivatives , Amides/chemistry , Cell Line, Tumor , Humans , Molecular Structure , Phosphoric Acids/chemistry , Ribose/chemistryABSTRACT
MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)-antimiR oligonucleotide complementary to the 5' end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake of the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and de-repression within 24 h of 199 liver mRNAs showing significant enrichment for miR-122 seed matches in their 3' UTRs. Expression profiling extended to 3 weeks after the last LNA-antimiR dose revealed that most of the changes in liver gene expression were normalized to saline control levels coinciding with normalized miR-122 and plasma cholesterol levels. Combined, these data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context.
Subject(s)
Gene Silencing , Liver/metabolism , MicroRNAs/antagonists & inhibitors , Oligonucleotides/pharmacology , RNA, Messenger/metabolism , Animals , Base Sequence , Female , Gene Expression Profiling , HeLa Cells , Humans , Liver/drug effects , Mice , MicroRNAs/chemistry , MicroRNAs/metabolism , Oligonucleotides/administration & dosage , Oligonucleotides/toxicity , Sequence Alignment , Up-RegulationABSTRACT
We have investigated the effects of different locked nucleic acid modified antisense mRNA antagonists against Survivin in a prostate cancer model. These mRNA antagonists were found to be potent inhibitors of Survivin expression at low nanomolar concentrations. Additionally there was a pronounced synergistic effect when combining the mRNA antagonists against Survivin with the chemotherapeutic Taxol. This effect was demonstrated at concentrations of antagonists far lower than any previously demonstrated, indicating the high potential of locked nucleic acid for therapeutic use. Further characterisations in vivo are ongoing.
Subject(s)
Antineoplastic Agents/pharmacology , Microtubule-Associated Proteins/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Oligonucleotides/chemistry , Prostatic Neoplasms/drug therapy , RNA, Antisense/pharmacology , RNA, Messenger/antagonists & inhibitors , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Humans , Inhibitor of Apoptosis Proteins , Male , RNA, Antisense/chemistry , RNA, Antisense/therapeutic use , SurvivinABSTRACT
Aptamers interacting with RNA hairpins through loop-loop (so-called kissing) interactions have been described as an alternative to antisense oligomers for the recognition of RNA hairpins. R06, an RNA aptamer, was previously shown to form a kissing complex with the TAR (trans-activating responsive) hairpin of HIV-1 RNA (Ducongé and Toulmé (1999) RNA 5, 1605). We derived a chimeric locked nucleic acid (LNA)/DNA aptamer from R06 that retains the binding properties of the originally selected R06 aptamer. We demonstrated that this LNA/DNA aptamer competes with a peptide of the retroviral protein Tat for binding to TAR, even though the binding sites of the two ligands do not overlap each other. This suggests that upon binding, the aptamer TAR adopts a conformation that is no longer appropriate for Tat association. In contrast, a LNA/DNA antisense oligomer, which exhibits the same binding constant and displays the same base-pairing potential as the chimeric aptamer, does not compete with Tat. Moreover, we showed that the LNA/DNA aptamer is a more specific TAR binder than the LNA/DNA antisense sequence. These results demonstrate the benefit of reading the three-dimensional shape of an RNA target rather than its primary sequence for the design of highly specific oligonucleotides.
Subject(s)
Aptamers, Nucleotide/chemistry , Gene Products, tat/chemistry , HIV Long Terminal Repeat , Nucleic Acid Conformation , Peptides/chemistry , RNA, Viral/chemistry , Base Pairing , Binding, Competitive , Oligodeoxyribonucleotides, Antisense/chemistryABSTRACT
One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera-TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets.
Subject(s)
HIV Long Terminal Repeat/genetics , HIV-1/genetics , Oligonucleotides, Antisense/metabolism , Oligoribonucleotides/metabolism , RNA, Viral/metabolism , RNA/metabolism , Base Sequence , Culture Media/pharmacology , Kinetics , Nucleic Acid Denaturation , Oligonucleotides , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/drug effects , Oligonucleotides, Antisense/genetics , Oligoribonucleotides/chemistry , Oligoribonucleotides/genetics , RNA/chemistry , RNA/drug effects , RNA/genetics , RNA, Viral/genetics , Substrate Specificity , Surface Plasmon Resonance , Temperature , ThermodynamicsABSTRACT
Full-length and 4 nucleotides truncated Locked Nucleic Acid (LNA) modifications of ISIS 3521 were compared for antisense properties in a cellular assay. ISIS 3521 is a 20-mer phosphorothioate designed to hybridise to human protein kinase C-alpha (PKC-alpha) mRNA and is currently submitted to clinical trials against cancer. We report that LNA can potentate this antisense oligo and retain the antisense potential with shorter oligos.
