Microglia-targeted inhibition of miR-17 via mannose-coated lipid nanoparticles improves pathology and behavior in a mouse model of Alzheimer's disease.

Neuroinflammation and accumulation of Amyloid Beta (Aß) accompanied by deterioration of special memory are hallmarks of Alzheimer's disease (AD). Effective preventative and treatment options for AD are still needed. Microglia in AD brains are characterized by elevated levels of microRNA-17 (miR-17), which is accompanied by defective autophagy, Aß accumulation, and increased inflammatory cytokine production. However, the effect of targeting miR-17 on AD pathology and memory loss is not clear. To specifically inhibit miR-17 in microglia, we generated mannose-coated lipid nanoparticles (MLNPs) enclosing miR-17 antagomir (Anti-17 MLNPs), which are targeted to mannose receptors readily expressed on microglia. We used a 5XFAD mouse model (AD) that recapitulates many AD-related phenotypes observed in humans. Our results show that Anti-17 MLNPs, delivered to 5XFAD mice by intra-cisterna magna injection, specifically deliver Anti-17 to microglia. Anti-17 MLNPs downregulated miR-17 expression in microglia but not in neurons, astrocytes, and oligodendrocytes. Anti-17 MLNPs attenuated inflammation, improved autophagy, and reduced Aß burdens in the brains. Additionally, Anti-17 MLNPs reduced the deterioration in spatial memory and decreased anxiety-like behavior in 5XFAD mice. Therefore, targeting miR-17 using MLNPs is a viable strategy to prevent several AD pathologies. This selective targeting strategy delivers specific agents to microglia without the adverse off-target effects on other cell types. Additionally, this approach can be used to deliver other molecules to microglia and other immune cells in other organs.

Co-treatment with miR-21-5p inhibitor and Aurora kinase inhibitor reversine suppresses breast cancer progression by targeting sprouty RTK signaling antagonist 2.

Numerous studies have reported the regulatory effects of miR-21-5p and reversine in human breast cancer (HBC). However, the mechanism of reversine and miR-21-5p has not been fully investigated in HBC. The aim of the current study was to assess the mechanism of action of reversine, with or without miR-21-5p, in HBC progression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot results confirmed the upregulation of miR-21-5p and downregulation of sprouty RTK signaling antagonist 2 (SPRY2) in HBC. Bioinformatics analysis and luciferase assay identified the correlation between miR-21-5p and SPRY2. Cell function experiment results indicated a decrease in migration, proliferation, and invasion of HBC cells treated with miR-21-5p inhibitor and reversine; however, an increase in apoptosis was observed in these cells. Apoptotic ability was more enhanced and migration, proliferation, and invasion were more impaired in HBC cells treated with both miR-21-5p inhibitor and reversine than in those treated individually with either inhibitors. SPRY2, downstream of miR-21-5p, participated in HBC progression with reversine. Overall, our study proved that combining the miR-21-5p inhibitor with reversine produced a synergistic effect by regulating SPRY2, thereby limiting HBC progression. This knowledge might offer insights into the clinical therapy of HBC.

A new player in the game: treatment with antagomiR-21a-5p significantly attenuates histological and echocardiographic effects of experimental autoimmune myocarditis.

AIMS: Myocarditis is associated with formidable symptoms and increased risk of adverse outcomes. Current approaches mostly rely on symptomatic treatments, warranting novel concepts for clinical practice. The aim of this study was to investigate the microRNA (miRNA) expression profile of Balb/c mice with experimental autoimmune myocarditis (EAM), choose a representative miRNA to antagonize after review of available literature and test its effects on myocardial inflammation in vitro and in vivo. METHODS AND RESULTS: Phase 1: EAM was induced in 12 male Balb/c mice, 10 animals served as controls. After sacrifice, next-generation sequencing (NGS) of the miRNA expression profile was performed. Based on these results, H9C2 cells and human ventricular cardiac fibroblasts exposed to lipopolysaccharide (LPS) were treated with the selected candidate antagomiR-21a-5p. Phase 2: EAM was induced in 48 animals. Thereof, 24 animals were either treated with antagomiR-21a-5p or negative control oligonucleotide in a nanoparticle formulation. Transthoracic echocardiography (TTE) was performed on Days 0, 7, 14, and 21. Histopathological examination was performed after sacrifice. Phase 1: EAM resulted in a significant up-regulation of 27 miRNAs, including miR-21a-5p (log2FC: 2.23, adj. P = 0.0026). Transfection with antagomiR-21a-5p resulted in a significant reduction of TNFα, IL-6, and collagen I in vitro. Phase 2: Treatment with antagomiR-21a-5p, formulated in polymeric nanoparticles for systemic injection, significantly attenuated myocardial inflammation (P = 0.001) and fibrosis (P = 0.013), as well as myocardial 'hypertrophy' on TTE. CONCLUSIONS: Silencing of miR-21a-5p results in a significant reduction of the expression of pro-inflammatory cytokines in vitro, as well as a significant attenuation of inflammation, fibrosis and echocardiographic effects of EAM in vivo.

Anti-MicroRNA-21 Oligonucleotide Loaded Spermine-Modified Acetalated Dextran Nanoparticles for B1 Receptor-Targeted Gene Therapy and Antiangiogenesis Therapy.

The use of nanoparticles (NPs) to deliver small inhibiting microRNAs (miRNAs) has shown great promise for treating cancer. However, constructing a miRNA delivery system that targets brain cancers, such as glioblastoma multiforme (GBM), remains technically challenging due to the existence of the blood-tumor barrier (BTB). In this work, a novel targeted antisense miRNA-21 oligonucleotide (ATMO-21) delivery system is developed for GBM treatment. Bradykinin ligand agonist-decorated spermine-modified acetalated dextran NPs (SpAcDex NPs) could temporarily open the BTB by activating G-protein-coupled receptors that are expressed in tumor blood vessels and tumor cells, which increase transportation to and accumulation in tumor sites. ATMO-21 achieves high loading in the SpAcDex NPs (over 90%) and undergoes gradual controlled release with the degradation of the NPs in acidic lysosomal compartments. This allows for cell apoptosis and inhibition of the expression of vascular endothelial growth factor by downregulating hypoxia-inducible factor (HIF-1α) protein. An in vivo orthotopic U87MG glioma model confirms that the released ATMO-21 shows significant therapeutic efficacy in inhibiting tumor growth and angiogenesis, demonstrating that agonist-modified SpAcDex NPs represent a promising strategy for GBM treatment combining targeted gene therapy and antiangiogenic therapy.

Magnolol attenuates the locomotor impairment, cognitive deficit, and neuroinflammation in Alzheimer's disease mice with brain insulin resistance via up-regulating miR-200c.

In this study, we aimed to investigate the effect of Magnolol on Alzheimer's disease (AD). After the model of streptozotocin-induced AD mice with brain insulin resistance was established, the mice were treated with Magnolol or miR-200c antagomiR. The abilities of ambulations, rearings, discrimination, spatial learning, and memory were evaluated by open-field test (OFT), novel object recognition (NOR), and morris water maze (MWM) tests. The levels of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), and miR-200c in the mice hippocampus were evaluated by enzyme-linked immunosorbent assay, Western blot, or Quantitative real-time Polymerase Chain Reaction. In AD mice model, streptozotocin induced the locomotor impairment and cognitive deficit, up-regulated levels of MDA, TNF-α, IL-6, and CRP, while down-regulated levels of GSH, SOD, and miR-200c. Magnolol increased the rearings numbers and discrimination index of AD mice in OFT and NOR tests. Magnolol increased the number of entries in the target quadrant and time spent in the target quadrant and decreased the escape latency of AD mice in the MWM test. Magnolol also down-regulated the levels of MDA, TNF-α, IL-6, and CRP, and up-regulated the levels of GSH, SOD, and miR-200c in the hippocampus tissues of AD mice. However, miR-200c antagomiR did the opposite and further offset the effects of the Magnolol on AD mice. Magnolol attenuated the locomotor impairment, cognitive deficit, and neuroinflammatory in AD mice with brain insulin resistance via up-regulating miR-200c.

AntimiR-132 Attenuates Myocardial Hypertrophy in an Animal Model of Percutaneous Aortic Constriction.

BACKGROUND: Pathological cardiac hypertrophy is a result of afterload-increasing pathologies including untreated hypertension and aortic stenosis. It features progressive adverse cardiac remodeling, myocardial dysfunction, capillary rarefaction, and interstitial fibrosis often leading to heart failure. OBJECTIVES: This study aimed to establish a novel porcine model of pressure-overload-induced heart failure and to determine the effect of inhibition of microribonucleic acid 132 (miR-132) on heart failure development in this model. METHODS: This study developed a novel porcine model of percutaneous aortic constriction by implantation of a percutaneous reduction stent in the thoracic aorta, inducing progressive remodeling at day 56 (d56) after pressure-overload induction. In this study, an antisense oligonucleotide specifically inhibiting miR-132 (antimiR-132), was regionally applied via intracoronary injection at d0 (percutaneous transverse aortic constriction induction) and d28. RESULTS: At d56, antimiR-132 treatment diminished cardiomyocyte cross-sectional area (188.9 ± 2.8 vs. 258.4 ± 9.0 µm2 in untreated hypertrophic hearts) and improved global cardiac function (ejection fraction 48.9 ± 1.0% vs. 36.1 ± 1.7% in control hearts). Moreover, at d56 antimiR-132-treated hearts displayed less increase of interstitial fibrosis compared with sham-operated hearts (Δsham 1.8 ± 0.5%) than control hearts (Δsham 10.8 ± 0.6%). Of note, cardiac platelet and endothelial cell adhesion molecule 1+ capillary density was higher in the antimiR-132-treated hearts (647 ± 20 cells/mm2) compared with in the control group (485 ± 23 cells/mm2). CONCLUSIONS: The inhibition of miR-132 is a valid strategy in prevention of heart failure progression in hypertrophic heart disease and may be developed as a treatment for heart failure of nonischemic origin.

LncRNA NEAT1 inhibition upregulates miR-16-5p to restrain the progression of sepsis-induced lung injury via suppressing BRD4 in a mouse model.

OBJECTIVE: Long non-coding RNAs (lncRNAs) are known to sponge microRNAs (miRNAs) to regulate biological processes. However, the role of nuclear paraspeckle assembly transcript 1 (NEAT1) binding miR-16-5p in sepsis-induced lung injury remains largely unknown. We aim to explore the effect of NEAT1 sponging miR-16-5p on sepsis-induced lung injury via regulating bromodomain containing 4 (BRD4). METHODS: A mouse model of sepsis-induced lung injury was established. Expression of NEAT1, miR-16-5p and BRD4 was determined. The pulmonary edema, myeloperoxidase (MPO) activity, pathological changes, levels of inflammatory factors, cell viability and apoptosis in mouse lung tissues were evaluated. The binding relationships between NEAT1 and miR-16-5p, and between miR-16-5p and BRD4 were confirmed. RESULTS: NEAT1 and BRD4 were upregulated while miR-16-5p was downregulated in sepsis-induced lung injury. NEAT1 inhibition or miR-16-5p elevation suppressed pulmonary edema, MPO activity, pathological changes, inflammation and apoptosis, and promoted cell viability in mouse lung tissues. NEAT1 bound with miR-16-5p and miR-16-5p targeted BRD4. CONCLUSION: NEAT1 inhibition upregulates miR-16-5p to repress the progression of sepsis-induced lung injury via downregulating BRD4.

Macrophage-derived EDA-A2 inhibits intestinal stem cells by targeting miR-494/EDA2R/ß-catenin signaling in mice.

The mucosa microenvironment is critical for intestinal stem cell self-renewal and reconstruction of the epithelial barrier in inflammatory bowel disease (IBD), where the mechanisms underlying cross-talk between intestinal crypts and the microenvironment remain unclear. Here, we firstly identified miR-494-3p as an important protector in colitis. miR-494-3p levels were decreased and negatively correlated with the severity in human IBD samples, as well as in colitis mice. In colitis crypts, a notable cytokine-cytokine receptor, miR-494-3p-targeted EDA2R and the ligand EDA-A2, suppressed colonic stemness and epithelial repair by inhibiting ß-catenin/c-Myc. In differentiated IECs, miR-494-3p inhibits macrophage recruitment, M1 activation and EDA-A2 secretion by targeting IKKß/NF-κB in colitis. A miR-494-3p agomir system notably ameliorated the severity of colonic colitis in vivo. Collectively, our findings uncover a miR-494-3p-mediated cross-talk mechanism by which macrophage-induced intestinal stem cell impairment aggravates intestinal inflammation.

Systemic delivery of antagomirs during blood-brain barrier disruption is disease-modifying in experimental epilepsy.

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.

Promotive role of microRNA­150 in hippocampal neurons apoptosis in vascular dementia model rats.

Cognitive impairment is one of the primary features of vascular dementia (VD). However, the specific mechanism underlying the regulation of cognition function in VD is not completely understood. The present study aimed to explore the effects of microRNA (miR)­150 on VD. To determine the effects of miR­150 on cognitive function and hippocampal neurons in VD model rats, rats were subjected to intracerebroventricular injections of miR­150 antagomiR. The Morris water maze test results demonstrated that spatial learning ability was impaired in VD model rats compared with control rats. Moreover, compared with antagomiR negative control (NC), miR­150 antagomiR alleviated cognitive impairment and enhanced memory ability in VD model rats. The triphenyltetrazolium chloride, Nissl staining and immunohistochemistry results further demonstrated that miR­150 knockdown improved the activity of hippocampal neurons in VD model rats compared with the antagomiR NC group. To validate the role of miR­150 in neurons in vitro, the PC12 cell line was used. The flow cytometry and Hoechst 33342/PI double staining results indicated that miR­150 overexpression significantly increased cell apoptosis compared with the mimic NC group. Moreover, the dual­luciferase reporter gene assay results indicated that miR­150 targeted HOXA1 and negatively regulated HOXA1 expression. Therefore, the present study indicated that miR­150 knockdown ameliorated VD symptoms by upregulating HOXA1 expression in vivo and in vitro.

Downregulation of microRNA-199a-5p alleviated lidocaine-induced sensory dysfunction and spinal cord myelin lesions in a rat model.

Lidocaine induces neurotoxicity in the spinal cord, but the underlying mechanisms remain unclear. In this study, we evaluated the effects of miR-199a-5p on 10 % lidocaine neurotoxicity. Increased expression of miR-199a-5p in the spinal cord of rats treated with 10 % lidocaine was assessed by qRT-PCR. Furthermore, after miR-199a-5p antagomir administration, the sensory dysfunction and myelin sheath lesions (evaluated by semithin sections stained with toluidine blue, electron microscopy, g-ratios and myelin thickness) induced by 10 % lidocaine were alleviated. Myelin regulatory factor (MYRF), a key molecule of myelin sheath development, was predicted to be a target gene of miR-199a-5p by the TargetScan and miRBase databases. MYRF and its downstream factors myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) were significantly decreased after intrathecal 10 % lidocaine administration. Moreover, these changes were reversed after miR-199a-5p antagomir administration. FISH-immunofluorescence showed coexpression of miR-199a-5p and MYRF in the spinal cord white matter of rats. A luciferase reporter assay further demonstrated the functional association between miR-199a-5p and MYRF. Overall, miR-199a-5p upregulation is involved in 10 % lidocaine-induced spinal cord toxicity through regulation of MYRF. Therefore, downregulating miR-199a-5p expression may be a potential strategy to ameliorate spinal cord neurotoxicity induced by 10 % lidocaine.

Down-regulation of miR-361-5p promotes the viability, migration and tube formation of endothelial progenitor cells via targeting FGF1.

Transplantion of bone marrow-derived endothelial progenitor cells (EPCs) may be a novel treatment for deep venous thrombosis (DVT). The present study probed into the role of microRNA (miR)-361-5p in EPCs and DVT recanalization. EPCs were isolated from male Sprague-Dawley (SD) rats and identified using confocal microscopy and flow cytometry. The viability, migration and tube formation of EPCs were examined using MTT assay, wound-healing assay and tube formation assay, respectively. Target gene and potential binding sites between miR-361-5p and fibroblast growth factor 1 (FGF1) were predicted by StarBase and confirmed by dual-luciferase reporter assay. Relative expressions of miR-361-5p and FGF1 were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. A DVT model in SD rats was established to investigate the role of EPC with miR-361-5p antagomir in DVT by Hematoxylin-Eosin (H&E) staining. EPC was identified as 87.1% positive for cluster of difference (CD)31, 2.17% positive for CD133, 85.6% positive for von Willebrand factor (vWF) and 94.8% positive for vascular endothelial growth factor receptor-2 (VEGFR2). MiR-361-5p antagomir promoted proliferation, migration and tube formation of EPCs and up-regulated FGF1 expression, thereby dissolving thrombus in the vein of DVT rats. FGF1 was the target of miR-361-5p, and overexpressed FGF1 reversed the effects of up-regulating miR-361-5p on suppressing EPCs. Down-regulation of miR-361-5p enhanced thrombus resolution in vivo and promoted EPC viability, migration and angiogenesis in vitro through targeting FGF1. Therefore, miR-361-5p may be a potential therapeutic target for DVT recanalization.

Yeast Cell wall Particle mediated Nanotube-RNA delivery system loaded with miR365 Antagomir for Post-traumatic Osteoarthritis Therapy via Oral Route.

Post-traumatic osteoarthritis (PTOA) is an acute injury-induced joint inflammation followed by a gradual degradation of articular cartilage. However, there is no FDA-approved Disease-Modifying Osteoarthritis Drug currently. Although gene therapy with microRNA can improve PTOA progression, there is no effective gene delivery vehicle for orally deliver therapeutics due to the harsh environment of the gastrointestinal tract. In this study, we investigated the effect of yeast cell wall particle (YCWP) mediated nanotube-RNA delivery system on PTOA therapy via oral route. Methods: Nontoxic and degradable AAT and miRNA365 antagomir was self-assembled into miR365 antagomir/AAT (NPs). Then NPs-YCWP oral drug delivery system was constructed by using NPs and non-pathogenic YCWP which can be specifically recognized by macrophages. Moreover, surgical destabilization of the medial meniscus induced PTOA mice model was established to evaluate the therapeutic effect of NPs-YCWP via oral route. Results: Compared with control group, NPs showed higher gene inhibition efficiency both in chondrogenic cell line and primary chondrocytes in vitro. Treatment of macrophages with fluorescently labeled NPs-YCWP, the results showed that NPs-YCWP was successfully engulfed by macrophages and participated in the regulation of gene expression in vitro. Under the protection of YCWP, miR365 antagomir/AAT passes through the gastrointestinal tract without degradation after oral administration. After NPs-YCWP therapy, the results of histological staining, gene and protein expression showed that miR365 antagomir/NPs-YCWP improved the symptom of PTOA. Conclusion: Here, we constructed a biodegradable drug delivery system based on non-pathogenic YCWP and nanotubes, which can be used for PTOA therapy via the oral route. It suggests a new gene therapy strategy with YCWP mediated oral nano drug delivery system may serve as a platform for joint degeneration treatment.

MicroRNA-181 regulates the development of Ossification of Posterior longitudinal ligament via Epigenetic Modulation by targeting PBX1.

Objectives: Ossification of the posterior longitudinal ligament (OPLL) presents as the development of heterotopic ossification in the posterior longitudinal ligament of the spine. The etiology of OPLL is genetically linked, as shown by its high prevalence in Asian populations. However, the molecular mechanism of the disease remains obscure. In this study, we explored the function and mechanism of OPLL-specific microRNAs. Methods: The expression levels of the ossification-related OPLL-specific miR-181 family were measured in normal or OPLL ligament tissues. The effect of miR-181a on the ossification of normal or pathogenic ligament cells was tested using real-time polymerase chain reaction (PCR), Western blot, alizarin red staining and alkaline phosphatase (ALP) staining. The candidate targets of miR-181 were screened using a dual luciferase reporter assay and functional analysis. The link between miR-181a and its target PBX1 was investigated using chromatin immunoprecipitation, followed by real-time PCR detection. Histological and immunohistochemical analysis as well as micro-CT scanning were used to evaluate the effects of miR-181 and its antagonist using both tip-toe-walking OPLL mice and in vivo bone formation assays. Results: Using bioinformatic analysis, we found that miR-181a-5p is predicted to play important roles in the development of OPLL. Overexpression of miR-181a-5p significantly increased the expression of ossification-related genes, staining level of alizarin red and ALP activity, while the inhibition of miR-181a-5p by treatment with an antagomir had the opposite effects. Functional analysis identified PBX1 as a direct target of miR-181a-5p, and we determined that PBX1 was responsible for miR-181a-5p's osteogenic phenotype. By chromatin immunoprecipitation assay, we found that miR-181a-5p controls ligament cell ossification by regulating PBX1-mediated modulation of histone methylation and acetylation levels in the promoter region of osteogenesis-related genes. Additionally, using an in vivo model, we confirmed that miR-181a-5p can substantially increase the bone formation ability of posterior ligament cells and cause increased osteophyte formation in the cervical spine of tip-toe-walking mice. Conclusions: Our data unveiled the mechanism by which the miR-181a-5p/PBX1 axis functions in the development of OPLL, and it revealed the therapeutic effects of the miR-181a-5p antagomir in preventing OPLL development both in vivo and in vitro. Our work is the first to demonstrate that microRNA perturbation could modulate the development of OPLL through epigenetic regulation.

Inhibition of miR-93 promotes interferon effector signaling to suppress influenza A infection by upregulating JAK1.

Type I interferons play a critical role in host defense against influenza virus infection. Interferon cascade induces the expression of interferon-stimulated genes then subsequently promotes antiviral immune responses. The microRNAs are important regulators of innate immunity, but microRNAs-mediated regulation of interferon cascade during influenza infection remains to be fully identified. Here we found influenza A virus (IAV) infection significantly inhibited miR-93 expression in alveolar epithelial type II cells through RIG-I/JNK pathway. IAV-induced downregulation of miR-93 was found to upregulate JAK1, the target of miR-93, and then feedback promote antiviral innate response by facilitating IFN effector signaling. Importantly, in vivo administration of miR-93 antagomiR markedly suppressed IAV infection, protecting mice form IAVs -associated death. Hence, the inducible downregulation of miR-93 feedback suppress IAV infection by strengthening IFN-JAK-STAT pathway via JAK1 upregulation, and in vivo inhibition of miR-93 bears considerable therapeutic potential for suppressing IAV infection.

Efficiency and Target Derepression of Anti-miR-92a: Results of a First in Human Study.

MicroRNA (miRNA) inhibition is a promising therapeutic strategy in several disease indications. MRG-110 is a locked nucleic acid-based antisense oligonucleotide that targets miR-92a-3p and experimentally was shown to have documented therapeutic effects on cardiovascular disease and wound healing. To gain first insights into the activity of anti-miR-92a in humans, we investigated miR-92a-3p expression in several blood compartments and assessed the effect of MRG-110 on target derepression. Healthy adults were randomly assigned (5:2) to receive a single intravenous dose of MRG-110 or placebo in one of seven sequential ascending intravenous dose cohorts ranging from 0.01 to 1.5 mg/kg body weight. MiR-92a-3p whole blood levels were time and dose dependently decreased with half-maximal inhibition of 0.27 and 0.31 mg/kg at 24 and 72 h after dosing, respectively. In the high-dose groups, >95% inhibition was detected at 24-72 h postinfusion and significant inhibition was observed for 2 weeks. Similar inhibitory effects were detected in isolated CD31+ cells, and miR-92a-3p expression was also inhibited in extracellular vesicles in the high-dose group. Target derepression was measured in whole blood and showed that ITGA5 and CD93 were increased at a dose of 1.5 mg/kg. Single-cell RNA sequencing of peripheral blood cells revealed a cell type-specific derepression of miR-92a targets. Together this study demonstrates that systemic infusion of anti-miR-92a efficiently inhibits miR-92a in the peripheral blood compartment and derepresses miR-92a targets in humans.

miR-134-5p inhibition reduces infarct-induced cardiomyocyte apoptosis via Creb1 upregulation.

BACKGROUND: Following the recent discovery that microRNA-134-5p (miR-134-5p) is elevated in the early stages of acute myocardial infarction (AMI), we examined the specific role of miR-134-5p in cardiomyocytes during AMI. METHODS: To study miR-134-5p's role in the context of AMI, we used a combination of in vitro experiments in H2O2-treated or hypoxic cardiomyocyte cell cultures as well as in vivo experiments in a murine model of AMI. RESULTS: H2O2- and hypoxia-induced cardiomyocyte injury upregulated miR-134-5p expression. miR-134-5p overexpression increased cardiomyocyte apoptosis, whereas miR-134-5p inhibition reduced cardiomyocyte apoptosis. We discovered that the transcription factor cAMP-responsive element binding protein 1 (Creb1) is a functional target of miR-134-5p responsible for regulating cardiomyocyte apoptosis. In vivo AMI resulted in the upregulation and downregulation of miR-134-5p and Creb1 in the infarct area, respectively. Circulating miR-134-5p levels were also increased at days 1 and 2 post-AMI. Modulation of myocardial miR-124-5p expression by intramyocardial injection of antagomiR-134-5p or agomiR-134-5p significantly affected cardiomyocyte apoptosis, infarct size, and cardiac function in vivo. CONCLUSIONS: miR-134-5p/Creb1 axis dysregulation plays a role in hypoxia- or oxidative stress-induced cardiomyocyte apoptosis as well as AMI. Circulating miR-134-5p may show promise as a biomarker for AMI or post-AMI cardiac dysfunction. Manipulating the miR-134-5p/Creb1 axis through either inhibition of miR-134-5p or overexpression of Creb1 may show promise as a novel therapeutic strategy to attenuate cardiac dysfunction following AMI.

In vivo miR-138-5p inhibition alleviates monocrotaline-induced pulmonary hypertension and normalizes pulmonary KCNK3 and SLC45A3 expression.

BACKGROUND: The pathogenesis of pulmonary arterial hypertension (PAH) involves many signalling pathways. MicroRNAs are potential candidates involved in simultaneously coordinating multiple genes under such multifactorial conditions. METHODS AND RESULTS: MiR-138-5p is overexpressed in pulmonary arterial smooth muscle cells (PASMCs) from PAH patients and in lungs from rats with monocrotaline-induced pulmonary hypertension (MCT-PH). MiR-138-5p is predicted to regulate the expression of the potassium channel KCNK3, whose loss is associated with the development and progression of PAH. We hypothesized that, in vivo, miR-138-5p inhibition would restore KCNK3 lung expression and subsequently alleviate PAH. Nebulization-based delivery of anti-miR-138-5p to rats with established MCT-PH significantly reduced the right ventricular systolic pressure and significantly improved the pulmonary arterial acceleration time (PAAT). These haemodynamic improvements were related to decrease pulmonary vascular remodelling, lung inflammation and pulmonary vascular cell proliferation in situ. In vivo inhibition of miR-138-5p restored KCNK3 mRNA expression and SLC45A3 protein expression in the lungs. CONCLUSIONS: We confirmed that in vivo inhibition of miR-138-5p reduces the development of PH in experimental MCT-PH. The possible curative mechanisms involve at least the normalization of lung KCNK3 as well as SLC45A3 expression.

Agonist of RORA Attenuates Nonalcoholic Fatty Liver Progression in Mice via Up-regulation of MicroRNA 122.

BACKGROUND & AIMS: Development of nonalcoholic steatohepatitis (NASH) is associated with reductions in hepatic microRNA122 (MIR122); the RAR related orphan receptor A (RORA) promotes expression of MIR122. Increasing expression of RORA in livers of mice increases expression of MIR122 and reduces lipotoxicity. We investigated the effects of a RORA agonist in mouse models of NASH. METHODS: We screened a chemical library to identify agonists of RORA and tested their effects on a human hepatocellular carcinoma cell line (Huh7). C57BL/6 mice were fed a chow or high-fat diet (HFD) for 4 weeks to induce fatty liver. Mice were given hydrodynamic tail vein injections of a MIR122 antagonist (antagomiR-122) or a control antagomiR once each week for 3 weeks while still on the HFD or chow diet, or intraperitoneal injections of the RORA agonist RS-2982 or vehicle, twice each week for 3 weeks. Livers, gonad white adipose, and skeletal muscle were collected and analyzed by reverse-transcription polymerase chain reaction, histology, and immunohistochemistry. A separate group of mice were fed an atherogenic diet, with or without injections of RS-2982 for 3 weeks; livers were analyzed by immunohistochemistry, and plasma was analyzed for levels of aminotransferases. We analyzed data from liver tissues from patients with NASH included in the RNA-sequencing databases GSE33814 and GSE89632. RESULTS: Injection of mice with antagomiR-122 significantly reduced levels of MIR122 in plasma, liver, and white adipose tissue; in mice on an HFD, antagomiR-122 injections increased fat droplets and total triglyceride content in liver and reduced ß-oxidation and energy expenditure, resulting in significantly more weight gain than in mice given the control microRNA. We identified RS-2982 as an agonist of RORA and found it to increase expression of MIR122 promoter activity in Huh7 cells. In mice fed an HFD or atherogenic diet, injections of RS-2982 increased hepatic levels of MIR122 precursors and reduced hepatic synthesis of triglycerides by reducing expression of biosynthesis enzymes. In these mice, RS-2982 significantly reduced hepatic lipotoxicity, reduced liver fibrosis, increased insulin resistance, and reduced body weight compared with mice injected with vehicle. Patients who underwent cardiovascular surgery had increased levels of plasma MIR122 compared to its levels before surgery; increased expression of plasma MIR122 was associated with increased levels of plasma free fatty acids and levels of RORA. CONCLUSIONS: We identified the compound RS-2982 as an agonist of RORA that increases expression of MIR122 in cell lines and livers of mice. Mice fed an HFD or atherogenic diet given injections of RS-2982 had reduced hepatic lipotoxicity, liver fibrosis, and body weight compared with mice given the vehicle. Agonists of RORA might be developed for treatment of NASH.

MiR-222 inhibition alleviates Staphylococcal Enterotoxin B-induced inflammatory acute lung injury by targeting Foxo3.

Acute lung injury (ALI) is a common acute and severe disease in clinical practice. Staphylococcal Enterotoxin B (SEB) is a superantigen that can cause inflammatory ALI. MiR-222 has been demonstrated to be upregulated in SEB-induced inflammatory ALI, but its exact roles and functions remain ill-defined. In this study, SEB exposure led to inflammatory ALI and high expression of miR-222 in model mice and lung infiltrating mononuclear cells, but the inflammatory response and high expression of miR-222 were restored in miR-222-/- mice. Moreover, we investigated the roles of miR-222 in vitro and observed that the concentrations of inflammatory cytokines and the expression of miR-222 were all elevated in SEB-activated splenocytes and miR-222 inhibition reversed the effects. Foxo3 was confirmed as a direct target of miR-222. Interestingly, SEB exposure led to a decrease of Foxo3 expression, and Foxo3 knockdown partially reversed the promotion of Foxo3 and the inhibition of inflammatory cytokines induced by miR-222 inhibitor in SEB-activated splenocytes. Our data indicated that miR-222 inhibition could alleviate SEB-induced inflammatory ALI by directly targeting Foxo3, shedding light on the potential therapeutic of miR-222 for SEB-induced inflammation in the lung.