MicroRNAs (miRNAs) are an abundant class of 20-23-nt long regulators of gene expression. The study of miRNA function in mice and potential therapeutic approaches largely depend on modified oligonucleotides. We recently demonstrated silencing miRNA function in mice using chemically modified and cholesterol-conjugated RNAs termed 'antagomirs'. Here, we further characterize the properties and function of antagomirs in mice. We demonstrate that antagomirs harbor optimized phosphorothioate modifications, require >19-nt length for highest efficiency and can discriminate between single nucleotide mismatches of the targeted miRNA. Degradation of different chemically protected miRNA/antagomir duplexes in mouse livers and localization of antagomirs in a cytosolic compartment that is distinct from processing (P)-bodies indicates a degradation mechanism independent of the RNA interference (RNAi) pathway. Finally, we show that antagomirs, although incapable of silencing miRNAs in the central nervous system (CNS) when injected systemically, efficiently target miRNAs when injected locally into the mouse cortex. Our data further validate the effectiveness of antagomirs in vivo and should facilitate future studies to silence miRNAs for functional analysis and in clinically relevant settings.

Diabetic patients with non-healing ulcers have a reduced expression of VEGF. Genetically diabetic mice have an altered expression pattern of VEGF and its receptor, VEGF receptor 2 (VEGFR-2). In diabetic wounds, the microRNAs, miR15b and miR200b, which respectively inhibit VEGF and VEGF-R2 mRNAs, are up-regulated, further affecting the impaired angiogenesis. We investigated whether anti-miRs directed toward miR15b and miR200b could improve wound repair in genetically diabetic mice.

MicroRNAs (miRNAs) are reported to involve in pathogenesis of temporal lobe epilepsy (TLE). miR-142-5p is found increased in TLE, but its role remains unknown. In the study, we established a mouse model of status epilepticus (SE) with pilocarpine and a cell model of TLE. Quantitative real-time PCR revealed an up-regulation of miR-142-5p and down-regulation of mitochondrial Rho 1 (Miro1) in the mouse mode of SE. Administration of miR-142-5p antagomirs via intracerebroventricular injection attenuated pilocarpine-induced SE and hippocampal damage, and alleviated mitochondrial dysfunction along with increased mitochondrial membrane potential and intracellular ATP and Ca (2+) levels. The expression of mitochondrial trafficking kinesin protein (Trak) 1 and Trak2 was up-regulated by inhibiting miR-142-5p. Antagomirs targeting miR-142-5p suppressed pilocarpine-induced oxidative stress as evidenced by decreased ROS generation and MPO activity, and increased SOD activity. Silencing miR-142-5p reduced neuronal death in pilocarpine-treated hippocampus and magnesium-free (MGF)-treated neurons. Inhibition of miR-142-5p decreased cytoplasmic Cytochrome C and increased mitochondrial Cytochrome C, reduced cleaved-caspase3 and Bax levels, and elevated Bcl2 in vivo and in vitro. Further, dual-luciferase assay verified Miro1 as a target of miR-142-5p, suggesting that miR-142-5p might function via targeting Mrio1. Depletion of Miro1 inhibited the protective effect of silencing miR-142-5p on hippocampal neurons in vitro. Taken together, down-regulation of miR-142-5p via targeting Miro1 inhibits neuronal death and mitochondrial dysfunction, and thus attenuates pilocarpine-induced SE, suggesting the potential involvement of miR-142-5p in the pathogenesis of TLE.

Depending on their physiological expression level, microRNAs (miRNA) address different target genes, thus have different biological functions. To identify these, the physiological expression has to be blocked. Here, we describe the use of inhibitory cholesterol-modified oligonucleotides (Antagomirs) to inhibit miRNAs selectively in primary human and murine T and B lymphocytes. Due to their lipophilic cholesterol tag Antagomirs enter primary lymphocytes efficiently and quantitatively. We show here that at concentrations of 0.125 to 1μM, Antagomirs selectively inhibit expression of their target miRNA up to 99.5% without affecting cell viability.

Oligonucleotide therapies offer precision treatments for a variety of neurological diseases, including epilepsy, but their deployment is hampered by the blood-brain barrier (BBB). Previous studies showed that intracerebroventricular injection of an antisense oligonucleotide (antagomir) targeting microRNA-134 (Ant-134) reduced evoked and spontaneous seizures in animal models of epilepsy. In this study, we used assays of serum protein and tracer extravasation to determine that BBB disruption occurring after status epilepticus in mice was sufficient to permit passage of systemically injected Ant-134 into the brain parenchyma. Intraperitoneal and intravenous injection of Ant-134 reached the hippocampus and blocked seizure-induced upregulation of miR-134. A single intraperitoneal injection of Ant-134 at 2 h after status epilepticus in mice resulted in potent suppression of spontaneous recurrent seizures, reaching a 99.5% reduction during recordings at 3 months. The duration of spontaneous seizures, when they occurred, was also reduced in Ant-134-treated mice. In vivo knockdown of LIM kinase-1 (Limk-1) increased seizure frequency in Ant-134-treated mice, implicating de-repression of Limk-1 in the antagomir mechanism. These studies indicate that systemic delivery of Ant-134 reaches the brain and produces long-lasting seizure-suppressive effects after systemic injection in mice when timed with BBB disruption and may be a clinically viable approach for this and other disease-modifying microRNA therapies.

MiRNAs have been shown to play a crucial role among lung cancer, pulmonary fibrosis, tuberculosis (TBC) infection, and bronchial hypersensitivity, thus including chronic obstructive pulmonary disease (COPD) and asthma. The oncogenic effect of several miRNAs has been recently ruled out. In order to act on miRNAs turnover, antagomiRs have been developed.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated cancer characterized by a high degree of recurrence, angiogenesis, and metastasis. The importance of alternative pro-angiogenesis pathways including viral factors has emerged after decades of directly targeting various signaling components. Using NPC as a model, we identified an essential oncogenic pathway underlying angiogenesis regulation that involves the inhibition of a tumor suppressor, Spry3, and its downstream targets by EBV-miR-BART10-5p (BART10-5p) and hsa-miR-18a (miR-18a). Overexpression of EBV-miR-BART10-5p and hsa-miR-18a strongly promotes angiogenesis in vitro and in vivo by regulating the expression of VEGF and HIF1-α in a Spry3-dependent manner. In vitro or in vivo treatment with iRGD-tagged exosomes containing antagomiR-BART10-5p and antagomiR-18a preferentially suppressed the angiogenesis and growth of NPC. Our findings first highlight the role of EBV-miR-BART10-5p and oncogenic hsa-miR-18a in NPC angiogenesis and also shed new insights into the clinical intervention and therapeutic strategies for nasopharyngeal carcinoma and other virus-associated tumors.

Lipid metabolic disorders have become a major global public health concern. Fatty liver and dyslipidemia are major manifestations of these disorders. Recently, MicroRNA-33 (miR-33), a post-transcriptional regulator of genes involved in cholesterol efflux and fatty acid oxidation, has been considered as a good therapeutic target for these disorders. However, the traditional methods of gene therapy impede their further clinical transformation into a mature treatment system. To counter this problem, in this study we used mesoporous silica nanoparticles (MSNs) as nanocarriers to deliver miR-33 antagomirs developing nanocomposites miR-MSNs. We observed that the hepatocellular uptake of miR-33 antagomirs increased by ∼5 times when they were delivered using miR-MSNs. The regulation effects of miR-MSNs on miR-33 and several genes involved in lipid metabolism were confirmed in L02 cells. In a high-fat diet fed mice, miR-33 intervention via miR-MSNs lowered the serum triglyceride levels remarkably by 18.9% and reduced hepatic steatosis. Thus, our results provide a proof-of-concept for a potential strategy to ameliorate lipid metabolic disorders.

In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory.

Studying microRNA networks during heart development is essential to obtain a better understanding of developmental defects and diseases associated with the heart and to identify novel opportunities for therapeutics. Here we highlight the advantages of chicken embryos as a vertebrate model for studying intermediate processes of heart development. Avians develop a four-chambered heart closely resembling human anatomy and they develop ex utero, which makes them easily accessible. Furthermore, embryos are available all year with a steady supply.

The effects of the existing anti-epileptic drugs are unsatisfactory to almost one third of epileptic patients. MiR-134 antagomirs prevent pilocarpine-induced status epilepticus. In this study, a lithium chloride-pilocarpine-induced status epilepticus model was established and treated with intracerebroventricular injection of antagomirs targeting miR-134 (Ant-134). The Ant-134 treatment significantly improved the performance of rats in Morris water maze tests, inhibited mossy fiber sprouting in the dentate gyrus, and increased the survival neurons in the hippocampal CA1 region. Silencing of miR-134 remarkably decreased malonaldehyde and 4-hydroxynonenal levels and increased superoxide dismutase activity in the hippocampus. The Ant-134 treatment also significantly increased the production of ATP and the activities of mitochondrial respiratory enzyme complexes and significantly decreased the reactive oxygen species generation in the hippocampus compared with the status epilepticus rats. Finally, the Ant-134 treatment remarkably downregulated the hippocampal expressions of autophagy-associated proteins Atg5, beclin-1 and light chain 3B. In conclusion, Ant-134 attenuates epilepsy via inhibiting oxidative stress, improving mitochondrial functions and regulating autophagy in the hippocampus.

MicroRNAs are post-transcriptional regulators of gene expression that are differentially regulated during development and in inflammatory diseases. A role for miRNAs in allergic asthma is emerging and further investigation is required to determine whether they may serve as potential therapeutic targets. We profiled miRNA expression in murine lungs from an ovalbumin-induced allergic airways disease model, and compared expression to animals receiving dexamethasone treatment and non-allergic controls. Our analysis identified 29 miRNAs that were significantly altered during allergic inflammation. Target prediction analysis revealed novel genes with altered expression in allergic airways disease and suggests synergistic miRNA regulation of target mRNAs. To assess the impacts of one induced miRNA on pathology, we targeted miR-155-5p using a specific antagomir. Antagomir administration successfully reduced miR-155-5p expression with high specificity, but failed to alter the disease phenotype. Interestingly, further investigation revealed that antagomir delivery has variable efficacy across different immune cell types, effectively targeting myeloid cell populations, but exhibiting poor uptake in lymphocytes. Our findings demonstrate that antagomir-based targeting of miRNA function in the lung is highly specific, but highlights cell-specificity as a key limitation to be considered for antagomir-based strategies as therapeutics.

Nasopharyngeal carcinoma (NPC) is a type of head and neck cancer with high incidence in China. The molecular mechanisms of vasculogenic mimicry (VM) and angiogenesis are not fully elucidated in NPC. More specially, it has seldomly been reported that Epstein-Barr virus-encoded miRNA can regulate VM and angiogenesis in NPC. The aim of this study was to investigate the function and molecular mechanism of a targeting exosome system (iRGD-exo-antagomiR) against VM and angiogenesis in NPC, and to provide new approaches for improving the comprehensive treatment of NPC.

Current anti-epileptic drugs (AEDs) act on a limited set of neuronal targets, are ineffective in a third of patients with epilepsy, and do not show disease-modifying properties. MicroRNAs are small noncoding RNAs that regulate levels of proteins by post-transcriptional control of mRNA stability and translation. MicroRNA-134 is involved in controlling neuronal microstructure and brain excitability and previous studies showed that intracerebroventricular injections of locked nucleic acid (LNA), cholesterol-tagged antagomirs targeting microRNA-134 (Ant-134) reduced evoked and spontaneous seizures in mouse models of status epilepticus. Translation of these findings would benefit from evidence of efficacy in non-status epilepticus models and validation in another species. Here, we report that electrographic seizures and convulsive behavior are strongly reduced in adult mice pre-treated with Ant-134 in the pentylenetetrazol model. Pre-treatment with Ant-134 did not affect the severity of status epilepticus induced by perforant pathway stimulation in adult rats, a toxin-free model of acquired epilepsy. Nevertheless, Ant-134 post-treatment reduced the number of rats developing spontaneous seizures by 86% in the perforant pathway stimulation model and Ant-134 delayed epileptiform activity in a rat ex vivo hippocampal slice model. The potent anticonvulsant effects of Ant-134 in multiple models may encourage pre-clinical development of this approach to epilepsy therapy.

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.

Glaucoma is a major cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP), which causes optic nerve damage and retinal ganglion cell death, is the primary risk factor for blindness in glaucoma patients. IOP is controlled by the balance between aqueous humor secretion from the ciliary body (CB) and its drainage through the trabecular meshwork (TM). How microRNAs (miRs) regulate IOP and glaucoma in vivo is largely unknown. Here we show that miR-143 and miR-145 expression is enriched in the smooth muscle and trabecular meshwork in the eye. Targeted deletion of miR-143/145 in mice results in significantly reduced IOP, consistent with an ~2-fold increase in outflow facilities. However, aqueous humor production in the same mice appears to be normal based on a microbeads-induced glaucoma model. Mechanistically, we found that miR-143/145 regulates actin dynamics and the contractility of TM cells, consistent with its regulation of actin-related protein complex (ARPC) subunit 2, 3, and 5, as well as myosin light chain kinase (MLCK) in these cells. Our data establish miR-143/145 as important regulators of IOP, which may have important therapeutic implications in glaucoma.

MicroRNAs (miRNAs) are short noncoding RNAs that down-regulate gene expression by silencing specific target mRNAs. While many miRNAs are transcribed from their own genes, nearly half map within introns of 'host' genes, the significance of which remains unclear. We report that transcriptional activation of apoptosis-associated tyrosine kinase (AATK), essential for neuronal differentiation, also generates miR-338 from an AATK gene intron that silences a family of mRNAs whose protein products are negative regulators of neuronal differentiation. We conclude that an intronic miRNA, transcribed together with the host gene mRNA, may serve the interest of its host gene by silencing a cohort of genes that are functionally antagonistic to the host gene itself.

Chronic cerebral hypoperfusion has been associated with cognitive impairment in dementias, such as Alzheimer's disease (AD) and vascular disease (VaD), the two most common neurodegenerative diseases in aged people. However, the effective therapeutic approaches for both AD and VaD are still missing. MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in the epigenetic regulation in many neurological disorders; the critical roles of miRNAderegulation had been implicated in both AD and VaD. In the current study, we reported that miR-9-5p is elevated in the serum and cerebrospinalfluid of patientswith VaD. The miR-9-5p wasalso increased in both the hippocampus and cortex of rats with 2-vessel occlusionsurgery. Furthermore, application ofmiR-9-5p antagomirs attenuated the memory impairments in rats with 2-vessel occlusion surgery both in the Morris water maze and inhibitory avoidance step-down tasks. Furthermore, miR-9-5p antagomirs reducedthe inhibition oflong-term potentiation and loss of dendritic spines in chronic cerebral hypoperfusionrats. Additionally, the cholinergic neuronal function was rescued by miR-9-5p antagomirs, as well as the neuronal loss and the oxidative stress. We concluded that miR-9-5p inhibition may be a potential therapeutic target for the memory impairments caused by chronic cerebral hypoperfusion.

MicroRNAs (miRNAs/miRs) are involved in a large number of biological functions and diseases, such as cancer, cardiovascular diseases, and diabetes. MiR-21 has been reported to target Sprouty homolog 1 (SPRY1), SMAD7, and PTEN. In this study, we examined the underlying role of miR-21 in the regulation of prorenin receptor (PRR)-mediated induction of vascular endothelial growth factor (VEGF) expression via targeting SMAD7, SPRY1, and PTEN in a hyperglycemic condition.

MicroRNAs (miRNAs) play critical regulatory roles in the pathogenesis of pulmonary fibrosis. The aim of this study was to explore whether miRNA antagomirs could serve as potential therapeutic agents in interstitial lung diseases.