31 results on '"Greene CM"'
Search Results
2. TGF-β1 Inhibition of ACE2 Mediated by miRNA Uncovers Novel Mechanism of SARS-CoV-2 Pathogenesis.
- Author
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Hejenkowska ED, Mitash N, Donovan JE, Chandra A, Bertrand C, De Santi C, Greene CM, Mu F, and Swiatecka-Urban A
- Subjects
- Humans, SARS-CoV-2 genetics, SARS-CoV-2 metabolism, Transforming Growth Factor beta1 genetics, Transforming Growth Factor beta1 metabolism, Angiotensin-Converting Enzyme 2 genetics, Angiotensin-Converting Enzyme 2 metabolism, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Pandemics, MicroRNAs genetics, COVID-19, Cystic Fibrosis
- Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, utilizes receptor binding domain (RBD) of spike glycoprotein to interact with angiotensin (Ang)-converting enzyme 2 (ACE2). Altering ACE2 levels may affect entry of SARS-CoV-2 and recovery from COVID-19. Decreased cell surface density of ACE2 leads to increased local levels of Ang II and may contribute to mortality resulting from acute lung injury and fibrosis during COVID-19. Studies published early during the COVID-19 pandemic reported that people with cystic fibrosis (PwCF) had milder symptoms, compared to people without CF. This finding was attributed to elevated ACE2 levels and/or treatment with the high efficiency CFTR modulators. Subsequent studies did not confirm these findings reporting variable effects of CFTR gene mutations on ACE2 levels. Transforming growth factor (TGF)-β signaling is essential during SARS-CoV-2 infection and dominates the chronic immune response in severe COVID-19, leading to pulmonary fibrosis. TGF-β1 is a gene modifier associated with more severe lung disease in PwCF but its effects on the COVID-19 course in PwCF is unknown. To understand whether TGF-β1 affects ACE2 levels in the airway, we examined miRNAs and their gene targets affecting SARS-CoV-2 pathogenesis in response to TGF-β1. Small RNAseq and micro(mi)RNA profiling identified pathways uniquely affected by TGF-β1, including those associated with SARS-CoV-2 invasion, replication, and the host immune responses. TGF-β1 inhibited ACE2 expression by miR-136-3p and miR-369-5p mediated mechanism in CF and non-CF bronchial epithelial cells. ACE2 levels were higher in two bronchial epithelial cell models expressing the most common CF-causing mutation in CFTR gene F508del, compared to controls without the mutation. After TGF-β1 treatment, ACE2 protein levels were still higher in CF, compared to non-CF cells. TGF-β1 prevented the modulator-mediated rescue of F508del-CFTR function while the modulators did not prevent the TGF-β1 inhibition of ACE2 levels. Finally, TGF-β1 reduced the interaction between ACE2 and the recombinant spike RBD by lowering ACE2 levels and its binding to RBD. Our data demonstrate novel mechanism whereby TGF-β1 inhibition of ACE2 in CF and non-CF bronchial epithelial cells may modulate SARS-CoV-2 pathogenicity and COVID-19 severity. By reducing ACE2 levels, TGF-β1 may decrease entry of SARS-CoV-2 into the host cells while hindering the recovery from COVID-19 due to loss of the anti-inflammatory and regenerative effects of ACE2. The above outcomes may be modulated by other, miRNA-mediated effects exerted by TGF-β1 on the host immune responses, leading to a complex and yet incompletely understood circuitry., (© 2023 The Author(s). Published by S. Karger AG, Basel.)
- Published
- 2023
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3. High-Throughput Identification of miRNA-Target Interactions in Melanoma Using miR-CATCHv2.0.
- Author
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Marranci A, D'Aurizio R, Rizzo M, Greene CM, and Poliseno L
- Subjects
- Cell Line, Tumor, Humans, Computational Biology, Gene Expression Profiling, High-Throughput Nucleotide Sequencing, Melanoma genetics, Melanoma metabolism, MicroRNAs genetics, MicroRNAs metabolism, RNA, Messenger genetics, RNA, Messenger metabolism
- Abstract
MicroRNAs (miRNAs) can regulate the expression of potentially every transcript in the cell, and the definition of miRNA-target interactions is crucial to understand their role in all biological processes. However, the identification of the miRNAs that target a specific mRNA remains a challenge. Here, we describe an innovative method called miR-CATCHv2.0 for the high-throughput identification of the miRNA species bound to an RNA of interest. We also describe how this method can overcome the limitations of the current computational and experimental methods available in this field.
- Published
- 2021
- Full Text
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4. Precise Targeting of miRNA Sites Restores CFTR Activity in CF Bronchial Epithelial Cells.
- Author
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De Santi C, Fernández Fernández E, Gaul R, Vencken S, Glasgow A, Oglesby IK, Hurley K, Hawkins F, Mitash N, Mu F, Raoof R, Henshall DC, Cutrona MB, Simpson JC, Harvey BJ, Linnane B, McNally P, Cryan SA, MacLoughlin R, Swiatecka-Urban A, and Greene CM
- Subjects
- Adult, Aminophenols pharmacology, Aminopyridines pharmacology, Benzodioxoles pharmacology, Bronchi cytology, Bronchi drug effects, Cells, Cultured, Child, Child, Preschool, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Drug Combinations, Drug Synergism, Epithelial Cells cytology, Epithelial Cells drug effects, Epithelial Cells metabolism, Female, Humans, Indoles pharmacology, Infant, Male, Middle Aged, Models, Biological, Nanoparticles, Oligonucleotides genetics, Polylactic Acid-Polyglycolic Acid Copolymer chemistry, Quinolones pharmacology, Bronchi metabolism, Cystic Fibrosis therapy, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, MicroRNAs genetics, Oligonucleotides pharmacology
- Abstract
MicroRNAs that are overexpressed in cystic fibrosis (CF) bronchial epithelial cells (BEC) negatively regulate CFTR and nullify the beneficial effects of CFTR modulators. We hypothesized that it is possible to reverse microRNA-mediated inhibition of CFTR using CFTR-specific target site blockers (TSBs) and to develop a drug-device combination inhalation therapy for CF. Lead microRNA expression was quantified in a series of human CF and non-CF samples and in vitro models. A panel of CFTR 3' untranslated region (UTR)-specific locked nucleic acid antisense oligonucleotide TSBs was assessed for their ability to increase CFTR expression. Their effects on CFTR activity alone or in combination with CFTR modulators were measured in CF BEC models. TSB encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles was assessed as a proof of principle of delivery into CF BECs. TSBs targeting the CFTR 3' UTR 298-305:miR-145-5p or 166-173:miR-223-3p sites increased CFTR expression and anion channel activity and enhanced the effects of ivacaftor/lumacaftor or ivacaftor/tezacaftor in CF BECs. Biocompatible PLGA-TSB nanoparticles promoted CFTR expression in primary BECs and retained desirable biophysical characteristics following nebulization. Alone or in combination with CFTR modulators, aerosolized CFTR-targeting TSBs encapsulated in PLGA nanoparticles could represent a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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5. Challenges facing microRNA therapeutics for cystic fibrosis lung disease.
- Author
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De Santi C and Greene CM
- Subjects
- Clinical Trials as Topic, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Humans, Mutation, Treatment Outcome, Cystic Fibrosis genetics, Cystic Fibrosis therapy, Genetic Therapy methods, MicroRNAs therapeutic use
- Published
- 2020
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6. Alpha-1 Antitrypsin-A Target for MicroRNA-Based Therapeutic Development for Cystic Fibrosis.
- Author
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Hunt AMD, Glasgow AMA, Humphreys H, and Greene CM
- Subjects
- Antagomirs pharmacology, Antagomirs therapeutic use, Cystic Fibrosis metabolism, Cystic Fibrosis therapy, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Humans, Leukocyte Elastase metabolism, MicroRNAs antagonists & inhibitors, Molecular Targeted Therapy, Up-Regulation, alpha 1-Antitrypsin metabolism, Cystic Fibrosis genetics, MicroRNAs genetics, alpha 1-Antitrypsin genetics
- Abstract
Cystic fibrosis (CF) is an autosomal recessive genetic disorder arising from mutations to the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. Disruption to normal ion homeostasis in the airway results in impaired mucociliary clearance, leaving the lung more vulnerable to recurrent and chronic bacterial infections. The CF lung endures an excess of neutrophilic inflammation, and whilst neutrophil serine proteases are a crucial part of the innate host defence to infection, a surplus of neutrophil elastase (NE) is understood to create a net destructive effect. Alpha-1 antitrypsin (A1AT) is a key antiprotease in the control of NE protease activity but is ineffective in the CF lung due to the huge imbalance of NE levels. Therapeutic strategies to boost levels of protective antiproteases such as A1AT in the lung remain an attractive research strategy to limit the damage from excess protease activity. microRNAs are small non-coding RNA molecules that bind specific cognate sequences to inhibit expression of target mRNAs. The inhibition of miRNAs which target the SERPINA1 (A1AT-encoding gene) mRNA represents a novel therapeutic approach for CF inflammation. This could involve the delivery of antagomirs that bind and sequester the target miRNA, or target site blockers that bind miRNA recognition elements within the target mRNA to prevent miRNA interaction. Therefore, miRNA targeted therapies offer an alternative strategy to drive endogenous A1AT production and thus supplement the antiprotease shield of the CF lung.
- Published
- 2020
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7. Plasma microRNA levels in male and female children with cystic fibrosis.
- Author
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Mooney C, McKiernan PJ, Raoof R, Henshall DC, Linnane B, McNally P, Glasgow AMA, and Greene CM
- Subjects
- Child, Child, Preschool, Cystic Fibrosis genetics, Female, Gene Ontology, Humans, Infant, Male, Prognosis, Reverse Transcriptase Polymerase Chain Reaction, Cystic Fibrosis blood, MicroRNAs blood, Sex Characteristics
- Abstract
A gender gap exists in cystic fibrosis (CF). Here we investigate whether plasma microRNA expression profiles differ between the sexes in CF children. MicroRNA expression was quantified in paediatric CF plasma (n = 12; six females; Age range:1-6; Median Age: 3; 9 p.Phe508del homo- or heterozygotes) using TaqMan OpenArray Human miRNA Panels. Principal component analysis indicated differences in male versus female miRNA profiles. The miRNA array analysis revealed two miRNAs which were significantly increased in the female samples (miR-885-5p; fold change (FC):5.07, adjusted p value: 0.026 and miR-193a-5p; FC:2.6, adjusted p value: 0.031), although only miR-885-5p was validated as increased in females using specific qPCR assay (p < 0.0001). Gene ontology analysis of miR-885-5p validated targets identified cell migration, motility and fibrosis as processes potentially affected, with RAC1-mediated signalling featuring significantly. There is a significant increase in miR-885-5p in plasma of females versus males with CF under six years of age.
- Published
- 2020
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8. The Estrogen-Induced miR-19 Downregulates Secretory Leucoprotease Inhibitor Expression in Monocytes.
- Author
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McKiernan PJ, Smith SGJ, Durham AL, Adcock IM, McElvaney NG, and Greene CM
- Subjects
- 3' Untranslated Regions genetics, Cells, Cultured, Down-Regulation, Estradiol metabolism, Estrogens metabolism, Female, Genes, myc genetics, Humans, Immunity, Innate, Male, Promoter Regions, Genetic genetics, Respiratory Tract Infections epidemiology, Respiratory Tract Infections metabolism, Secretory Leukocyte Peptidase Inhibitor genetics, Sex Characteristics, MicroRNAs genetics, Monocytes metabolism, Respiratory Tract Infections genetics, Secretory Leukocyte Peptidase Inhibitor metabolism, Sex Factors
- Abstract
Compared to females, males are more susceptible to acute viral and other respiratory tract infections that display greater severity and higher mortality. In contrast, females tend to fare worse with chronic inflammatory diseases. Circulating 17β-estradiol (E2) is a female-specific factor that may influence the progression of human lung diseases. Here we hypothesize that E2 modulates the inflammatory response of monocytes through microRNA (miRNA)-based modulation of secretory leucoprotease inhibitor (SLPI), an antiprotease with immunomodulatory effects. Monocytic cells were treated ± E2, and differentially expressed miRNAs were identified using PCR profiling. Cells were transfected with miRNA mimics or antimiRs and SLPI mRNA and protein levels were quantified. Luciferase activity assay using wildtype and ΔmiR-19a/b-SLPI3'UTR reporter constructs and chromatin immunoprecipitation on E2-treated monocytes were performed. E2 downregulated SLPI and upregulated miR-19 expression in monocytes. Transfection with premiR-19b reduced SLPI mRNA and protein levels and this effect was abrogated using antimiRs against miR-19b. miR-19b directly binds the SLPI 3'UTR. The mechanism responsible for E2-mediated upregulation of miR-19 occurs via increased MIR17HG promoter activity mediated by c-MYC. Overall E2 decreases SLPI expression in human monocytic cells, via changes in miRNA expression and highlights the potential for estrogen to modulate the innate immune system., (© 2019 The Author(s) Published by S. Karger AG, Basel.)
- Published
- 2020
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9. Transforming Growth Factor-β1 Selectively Recruits microRNAs to the RNA-Induced Silencing Complex and Degrades CFTR mRNA under Permissive Conditions in Human Bronchial Epithelial Cells.
- Author
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Mitash N, Mu F, Donovan JE, Myerburg MM, Ranganathan S, Greene CM, and Swiatecka-Urban A
- Subjects
- Bronchi cytology, Cells, Cultured, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Gene Silencing, Humans, MicroRNAs genetics, Respiratory Mucosa drug effects, Bronchi metabolism, Cystic Fibrosis Transmembrane Conductance Regulator genetics, MicroRNAs metabolism, RNA Stability, Respiratory Mucosa metabolism, Transforming Growth Factor beta pharmacology
- Abstract
Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (
CFTR ) gene lead to cystic fibrosis (CF). The most common mutation F508del inhibits folding and processing of CFTR protein. FDA-approved correctors rescue the biosynthetic processing of F508del-CFTR protein, while potentiators improve the rescued CFTR channel function. Transforming growth factor (TGF-β1), overexpressed in many CF patients, blocks corrector/potentiator rescue by inhibiting CFTR mRNA in vitro. Increased TGF-β1 signaling and acquired CFTR dysfunction are present in other lung diseases. To study the mechanism of TGF-β1 repression of CFTR, we used molecular, biochemical, and functional approaches in primary human bronchial epithelial cells from over 50 donors. TGF-β1 destabilized CFTR mRNA in cells from lungs with chronic disease, including CF, and impaired F508del-CFTR rescue by new-generation correctors. TGF-β1 increased the active pool of selected micro(mi)RNAs validated as CFTR inhibitors, recruiting them to the RNA-induced silencing complex (RISC). Expression of F508del-CFTR globally modulated TGF-β1-induced changes in the miRNA landscape, creating a permissive environment required for degradation of F508del-CFTR mRNA. In conclusion, TGF-β1 may impede the full benefit of corrector/potentiator therapy in CF patients. Studying miRNA recruitment to RISC under disease-specific conditions may help to better characterize the miRNAs utilized by TGF-β1 to destabilize CFTR mRNA.- Published
- 2019
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10. Systematic evaluation of the microRNAome through miR-CATCHv2.0 identifies positive and negative regulators of BRAF -X1 mRNA.
- Author
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Marranci A, D'Aurizio R, Vencken S, Mero S, Guzzolino E, Rizzo M, Pitto L, Pellegrini M, Chiorino G, Greene CM, and Poliseno L
- Subjects
- Humans, MicroRNAs genetics, Protein Isoforms genetics, Protein Isoforms metabolism, RNA Stability genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Reproducibility of Results, MicroRNAs metabolism, Proto-Oncogene Proteins B-raf genetics, Software
- Abstract
Here we present miR-CATCHv2.0, an implemented experimental method that allows the identification of the microRNA species directly bound to an RNA of interest. After cross-linking of microRNA::RNA::Ago2 complexes using formaldehyde, the RNA is fragmented using sonication and then subjected to affinity purification using two sets of biotinylated tiling probes (ODD and EVEN). Finally, enriched microRNA species are retrieved by means of small RNA sequencing coupled with an ad hoc analytical workflow. In BRAFV600E mutant A375 melanoma cells, miR-CATCHv2.0 allowed us to identify 20 microRNAs that target X1, the most abundant isoform of BRAF mRNA. These microRNAs fall into different functional classes, according to the effect that they exert (decrease/increase in BRAFV600E mRNA and protein levels) and to the mechanism they use to achieve it (destabilization/stabilization of X1 mRNA or decrease/increase in its translation). microRNA-induced variations in BRAFV600E protein levels are most of the times coupled to consistent variations in pMEK levels, in melanoma cell proliferation in vitro and in sensitivity to the BRAF inhibitor vemurafenib in a xenograft model in zebrafish. However, microRNAs exist that uncouple the degree of activation of the ERK pathway from the levels of BRAFV600E protein. Our study proposes miR-CATCHv2.0 as an effective tool for the identification of direct microRNA-target interactions and, by using such a tool, unveils the complexity of the post-transcriptional regulation to which BRAFV600E and the ERK pathway are subjected in melanoma cells.
- Published
- 2019
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11. X Chromosome-encoded MicroRNAs Are Functionally Increased in Cystic Fibrosis Monocytes.
- Author
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McKiernan PJ, Molloy KP, Cryan SA, McElvaney NG, and Greene CM
- Subjects
- Adult, Female, Humans, Male, Polymerase Chain Reaction, Young Adult, Chromosomes, Human, X genetics, Cystic Fibrosis genetics, MicroRNAs genetics, Monocytes
- Published
- 2018
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12. Alpha-1 antitrypsin augmentation therapy decreases miR-199a-5p, miR-598 and miR-320a expression in monocytes via inhibition of NFκB.
- Author
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Hassan T, de Santi C, Mooney C, McElvaney NG, and Greene CM
- Subjects
- Adult, Case-Control Studies, Cells, Cultured, Female, Gene Expression Profiling, Gene Expression Regulation, Humans, Male, Middle Aged, Monocytes drug effects, Monocytes pathology, NF-kappa B genetics, NF-kappa B metabolism, Serine Proteinase Inhibitors, alpha 1-Antitrypsin Deficiency drug therapy, alpha 1-Antitrypsin Deficiency metabolism, MicroRNAs genetics, Monocytes metabolism, NF-kappa B antagonists & inhibitors, Trypsin Inhibitors therapeutic use, alpha 1-Antitrypsin Deficiency genetics
- Abstract
Alpha-1 antitrypsin (AAT) augmentation therapy involves infusion of plasma-purified AAT to AAT deficient individuals. Whether treatment affects microRNA expression has not been investigated. This study's objectives were to evaluate the effect of AAT augmentation therapy on altered miRNA expression in monocytes and investigate the mechanism. Monocytes were isolated from non-AAT deficient (MM) and AAT deficient (ZZ) individuals, and ZZs receiving AAT. mRNA (qRT-PCR, microarray), miRNA (miRNA profiling, qRT-PCR), and protein (western blotting) analyses were performed. Twenty one miRNAs were differentially expressed 3-fold between ZZs and MMs. miRNA validation studies demonstrated that in ZZ monocytes receiving AAT levels of miR-199a-5p, miR-598 and miR-320a, which are predicted to be regulated by NFκB, were restored to levels similar to MMs. Validated targets co-regulated by these miRNAs were reciprocally increased in ZZs receiving AAT in vivo and in vitro. Expression of these miRNAs could be increased in ZZ monocytes treated ex vivo with an NFκB agonist and decreased by NFκB inhibition. p50 and p65 mRNA and protein were significantly lower in ZZs receiving AAT than untreated ZZs. AAT augmentation therapy inhibits NFκB and decreases miR-199a-5p, miR-598 and miR-320a in ZZ monocytes. These NFκB-inhibitory properties may contribute to the anti-inflammatory effects of AAT augmentation therapy.
- Published
- 2017
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13. Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing.
- Author
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De Santi C, Vencken S, Blake J, Haase B, Benes V, Gemignani F, Landi S, and Greene CM
- Subjects
- Cell Line, Tumor, Cell Proliferation genetics, GPI-Linked Proteins metabolism, High-Throughput Nucleotide Sequencing, Humans, Mesothelin, MicroRNAs metabolism, GPI-Linked Proteins genetics, Gene Expression Regulation, Neoplastic, MicroRNAs genetics
- Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA expression mainly by silencing target transcripts via binding to miRNA recognition elements (MREs) in the 3'untranslated region (3'UTR). The identification of bona fide targets is challenging for researchers working on the functional aspect of miRNAs. Recently, we developed a method (miR-CATCH) based on biotinylated DNA antisense oligonucleotides that capture the mRNA of interest and facilitates the characterisation of miRNAs::mRNA interactions in a physiological cellular context. Here, the miR-CATCH technique was applied to the mesothelin (MSLN) gene and coupled with next generation sequencing (NGS), to identify miRNAs that regulate MSLN mRNA and that may be responsible for its increased protein levels found in malignant pleural mesothelioma (MPM). Biotinylated MSLN oligos were employed to isolate miRNA::MSLN mRNA complexes from a normal cell line (Met-5A) which expresses low levels of MSLN. MiRNAs targeting the MSLN mRNA were identified by NGS and miR-21-5p and miR-100-5p were selected for further validation analyses. MiR-21-5p was shown to be able to modulate MSLN expression in miRNA mimic experiments in a panel of malignant and non-malignant cell lines. Further miRNA inhibitor experiments and luciferase assays in Mero-14 cells validated miR-21-5p as a true regulator of MSLN. Moreover, in vitro experiments showed that treatment with miR-21-5p mimic reduced proliferation of MPM cell lines. Altogether, this work shows that the miR-CATCH technique, coupled with NGS and in vitro validation, represents a reliable method to identify native miRNA::mRNA interactions. MiR-21-5p is suggested as novel regulator of MSLN with a possible functional role in cellular growth., Competing Interests: This work was supported by two grants: Science Foundation Ireland 12/TIDA/B2265 and Ministero della Salute-Bando Ricerca Finalizzata 2009 (RF-2009-1529895). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was also possible due to the PI being awarded the European Respiratory Society Rare Disease Award 2013 which was sponsored by GlaxoSmithKline. This commercial entity had no role in the study design; collection, analysis, and interpretation of data; writing of the paper; and/or decision to submit for publication. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
- Published
- 2017
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14. microRNA regulatory circuits in a mouse model of inherited retinal degeneration.
- Author
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Palfi A, Hokamp K, Hauck SM, Vencken S, Millington-Ward S, Chadderton N, Carrigan M, Kortvely E, Greene CM, Kenna PF, and Farrar GJ
- Subjects
- Animals, Chromatography, Liquid, Disease Models, Animal, Gene Expression Profiling, Mice, Tandem Mass Spectrometry, Gene Regulatory Networks, MicroRNAs analysis, Retina pathology, Retinal Degeneration pathology
- Abstract
miRNA dysregulation is a hallmark of many neurodegenerative disorders, including those involving the retina. Up-regulation of miR-1/133 and miR-142, and down-regulation of miR-183/96/182 has been described in the RHO-P347S mouse retina, a model for a common form of inherited blindness. High-throughput LC-MS/MS was employed to analyse the protein expression of predicted targets for these miRNAs in RHO-P347S mouse retinas; 133 potential target genes were identified. Pathway over-representation analysis suggests G-protein signaling/visual transduction, and synaptic transmission for miR-1, and transmembrane transport, cell-adhesion, signal transduction and apoptosis for miR-183/96/182 as regulated functions in retina. Validation of miRNA-target mRNA interactions for miR-1, miR-96/182 and miR-96 targeting Ctbp2, Rac1 and Slc6a9, respectively, was demonstrated in vitro. In vivo interaction of miR-183/96/182 and Rac1 mRNA in retina was confirmed using miR-CATCH. Additional miRNAs (including miR-103-3p, miR-9-5p) were both predicted to target Rac1 mRNA and enriched by Rac1-miR-CATCH. Other Rac1-miR-CATCH-enriched miRNAs (including miR-125a/b-5p, miR-378a-3p) were not predicted to target Rac1. Furthermore, levels of ~25% of the retinal Rac1 interactors were determined by LC-MS/MS; expression of Rap1gds1 and Cav1 was elevated. Our data suggest significant utilisation of miRNA-based regulation in retina. Possibly more than 30 miRNAs interact with Rac1 in retina, targeting both UTRs and coding regions.
- Published
- 2016
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15. High-throughput profiling for discovery of non-coding RNA biomarkers of lung disease.
- Author
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McKiernan PJ and Greene CM
- Subjects
- Animals, Biomarkers blood, Humans, MicroRNAs genetics, Polymerase Chain Reaction methods, RNA, Long Noncoding genetics, High-Throughput Screening Assays methods, Lung Diseases blood, MicroRNAs blood, Molecular Diagnostic Techniques methods, RNA, Long Noncoding blood
- Abstract
In respiratory medicine there is a need for clinical biomarkers for diagnosis, prognosis and assessment of response to therapy. Noncoding RNA (ncRNA) is expressed in all human cells; two major classes--long ncRNA and microRNA--are detectable extracellularly in the circulation and other biofluids. Altered ncRNA expression is associated with lung disease; collectively this indicates that ncRNA represents a potential biomarker class. This article presents and compares existing platforms for detection and quantification of ncRNA, specifically hybridization, qRT-PCR and RNA sequencing, and outlines methods for data interpretation and normalization. Each approach has merits and shortcomings, which can affect the choice of method when embarking on a biomarker study. Biomarker properties and pre-analytical considerations for ncRNA profiling are also presented. Since a variety of profiling approaches are available, careful study and experimental design are important. Finally, challenges and goals for reliable, standardized high-throughput ncRNA profiling in biofluids as lung disease biomarkers are reviewed.
- Published
- 2016
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16. Toll-Like Receptors in Cystic Fibrosis: Impact of Dysfunctional microRNA on Innate Immune Responses in the Cystic Fibrosis Lung.
- Author
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Vencken SF and Greene CM
- Subjects
- Animals, Gene Expression Regulation, Humans, Inflammation, Signal Transduction, Toll-Like Receptors genetics, Cystic Fibrosis immunology, Immunity, Innate genetics, Infections immunology, Lung immunology, MicroRNAs genetics, Respiratory Mucosa immunology, Toll-Like Receptors metabolism
- Abstract
Toll-like receptors (TLRs) are a class of pattern recognition receptors that are particularly expressed in the sentinel and epithelial cells in the body, including the lung. They are central players in the innate immune system in response to microbial infection, and are the triggers of a complex pathway network that both promotes the inflammatory response and influences the adaptive immune response. These pathways are transiently and finely tuned by cellular factors, including a cell's microRNA response program. MicroRNAs are small, non-coding RNAs that specifically regulate gene expression. In this article, we review the disease-specific microRNA regulatory network of cystic fibrosis, a debilitating and ultimately fatal disease and, specifically, its effect on TLR signalling., (© 2016 S. Karger AG, Basel.)
- Published
- 2016
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17. miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production.
- Author
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Oglesby IK, Vencken SF, Agrawal R, Gaughan K, Molloy K, Higgins G, McNally P, McElvaney NG, Mall MA, and Greene CM
- Subjects
- Adult, Animals, Bronchi cytology, Bronchoalveolar Lavage Fluid, Cell Count, Cell Line, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Female, Humans, Interleukin-8 genetics, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, MicroRNAs genetics, Middle Aged, Young Adult, Cystic Fibrosis immunology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Epithelial Cells metabolism, Interleukin-8 metabolism, MicroRNAs metabolism, Neutrophil Infiltration
- Abstract
Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis. IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in βENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured. miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, βENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o(-) tracheal, CFBE41o(-) and/or IB3 bronchial epithelial cells. These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases., (Copyright ©ERS 2015.)
- Published
- 2015
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18. Non-coding RNA as lung disease biomarkers.
- Author
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Vencken SF, Greene CM, and McKiernan PJ
- Subjects
- Humans, Lung Diseases genetics, Biomarkers, Tumor metabolism, Lung Diseases diagnosis, Lung Diseases metabolism, MicroRNAs metabolism, RNA, Long Noncoding metabolism
- Abstract
Biomarkers are quantifiable indicators of disease. These surrogates should be specific, sensitive, predictive, robust and easily accessible. A major class of RNA described as non-coding RNA fulfils many of these criteria, and recent studies have demonstrated that the two major subclasses of non-coding RNA, long non-coding RNA and, in particular, microRNA are promising potential biomarkers. The ability to detect non-coding RNAs in biofluids has highlighted their usefulness as non-invasive markers of lung disease. Because expression of specific non-coding RNAs is altered in many lung diseases and their levels in the circulation often reflect the changes in expression of their lung-specific counterparts, exploiting these biomolecules as diagnostic tools seems an obvious goal. New technology is driving developments in this area and there has been significant recent progress with respect to lung cancer diagnostics. The non-coding RNA biomarker field represents a clear example of modern-day bench-to-bedside research., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)
- Published
- 2015
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19. MicroRNA Dysregulation in Cystic Fibrosis.
- Author
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McKiernan PJ and Greene CM
- Subjects
- Cystic Fibrosis Transmembrane Conductance Regulator physiology, Endoplasmic Reticulum Stress, Gene Expression Regulation, Humans, Immunity, Innate, Toll-Like Receptors physiology, Cystic Fibrosis genetics, MicroRNAs physiology
- Abstract
The cystic fibrosis lung is a complex milieu comprising multiple factors that coordinate its physiology. MicroRNAs are regulatory factors involved in most biological processes and it is becoming increasingly clear that they play a key role in the development and manifestations of CF lung disease. These small noncoding RNAs act posttranscriptionally to inhibit protein production. Their involvement in the pathogenesis of CF lung disease stems from the fact that their expression is altered in vivo in the CF lung due to intrinsic and extrinsic factors; to date defective chloride ion conductance, endoplasmic reticulum stress, inflammation, and infection have been implicated in altering endogenous miRNA expression in this setting. Here, the current state-of-the-art and biological consequences of altered microRNA expression in cystic fibrosis are reviewed.
- Published
- 2015
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20. Developmental control of CFTR: from bioinformatics to novel therapeutic approaches.
- Author
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Greene CM and Hartl D
- Subjects
- Animals, Female, Humans, Male, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Gene Expression Regulation, Developmental, MicroRNAs metabolism
- Published
- 2015
- Full Text
- View/download PDF
21. miR-CATCH: microRNA capture affinity technology.
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Vencken S, Hassan T, McElvaney NG, Smith SG, and Greene CM
- Subjects
- Base Pairing, Base Sequence, Cell Line, Humans, MicroRNAs chemistry, MicroRNAs genetics, Molecular Sequence Data, Oligonucleotides, Antisense chemical synthesis, Oligonucleotides, Antisense genetics, RNA, Messenger chemistry, RNA, Messenger genetics, MicroRNAs isolation & purification, Nucleic Acid Hybridization methods, RNA, Messenger isolation & purification
- Abstract
Several experimental methods exist to explore the microRNA (miRNA) regulome. These methods almost exclusively focus on multiple targets bound to a single, or perhaps a few miRNAs of interest. Here, we describe a microRNA capture affinity technology (miR-CATCH) which uses an affinity capture oligonucleotide to co-purify a single target messenger RNA (mRNA) together with all its endogenously bound miRNAs. This bench-top method is similar to RNA immunoprecipitation (RIP) and provides an experimental alternative to computational miRNA target prediction.
- Published
- 2015
- Full Text
- View/download PDF
22. miR-31 dysregulation in cystic fibrosis airways contributes to increased pulmonary cathepsin S production.
- Author
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Weldon S, McNally P, McAuley DF, Oglesby IK, Wohlford-Lenane CL, Bartlett JA, Scott CJ, McElvaney NG, Greene CM, McCray PB Jr, and Taggart CC
- Subjects
- Adolescent, Biomarkers metabolism, Blotting, Western, Bronchoalveolar Lavage Fluid microbiology, Cell Line, Child, Child, Preschool, Cystic Fibrosis metabolism, Cystic Fibrosis microbiology, Down-Regulation, Enzyme-Linked Immunosorbent Assay, Humans, Infant, Peptide Hydrolases metabolism, Pseudomonas Infections complications, Pseudomonas Infections diagnosis, Pseudomonas Infections metabolism, Pseudomonas aeruginosa isolation & purification, Respiratory Mucosa microbiology, Reverse Transcriptase Polymerase Chain Reaction, Bronchoalveolar Lavage Fluid chemistry, Cathepsins metabolism, Cystic Fibrosis genetics, Interferon Regulatory Factor-1 metabolism, MicroRNAs metabolism, Respiratory Mucosa metabolism
- Abstract
Rationale: Cathepsin S (CTSS) activity is increased in bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis (CF). This activity contributes to lung inflammation via degradation of antimicrobial proteins, such as lactoferrin and members of the β-defensin family., Objectives: In this study, we investigated the hypothesis that airway epithelial cells are a source of CTSS, and mechanisms underlying CTSS expression in the CF lung., Methods: Protease activity was determined using fluorogenic activity assays. Protein and mRNA expression were analyzed by ELISA, Western blotting, and reverse-transcriptase polymerase chain reaction., Measurements and Main Results: In contrast to neutrophil elastase, CTSS activity was detectable in 100% of CF BAL fluid samples from patients without Pseudomonas aeruginosa infection. In this study, we identified epithelial cells as a source of pulmonary CTSS activity with the demonstration that CF airway epithelial cells express and secrete significantly more CTSS than non-CF control cells in the absence of proinflammatory stimulation. Furthermore, levels of the transcription factor IRF-1 correlated with increased levels of its target gene CTSS. We discovered that miR-31, which is decreased in the CF airways, regulates IRF-1 in CF epithelial cells. Treating CF bronchial epithelial cells with a miR-31 mimic decreased IRF-1 protein levels with concomitant knockdown of CTSS expression and secretion., Conclusions: The miR-31/IRF-1/CTSS pathway may play a functional role in the pathogenesis of CF lung disease and may open up new avenues for exploration in the search for an effective therapeutic target.
- Published
- 2014
- Full Text
- View/download PDF
23. miR-199a-5p silencing regulates the unfolded protein response in chronic obstructive pulmonary disease and α1-antitrypsin deficiency.
- Author
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Hassan T, Carroll TP, Buckley PG, Cummins R, O'Neill SJ, McElvaney NG, and Greene CM
- Subjects
- Adult, Asymptomatic Diseases, Biomarkers metabolism, Blotting, Western, Cytokines metabolism, Endoplasmic Reticulum Chaperone BiP, Female, Gene Expression Profiling, Humans, Male, Methylation, Middle Aged, Monocytes metabolism, Phenotype, Polymerase Chain Reaction, Pulmonary Disease, Chronic Obstructive complications, Pulmonary Disease, Chronic Obstructive metabolism, Sequence Analysis, RNA, Stress, Physiological, Unfolded Protein Response physiology, Up-Regulation, alpha 1-Antitrypsin Deficiency complications, alpha 1-Antitrypsin Deficiency metabolism, Gene Silencing, MicroRNAs metabolism, Pulmonary Disease, Chronic Obstructive genetics, Unfolded Protein Response genetics, alpha 1-Antitrypsin Deficiency genetics
- Abstract
Rationale: Retention of abnormal α1-antitrypsin (AAT) activates the unfolded protein response in AAT-deficient monocytes. The regulatory role of microRNAs (miRNAs) in unfolded protein responses and chronic obstructive pulmonary disease pathogenesis has not been investigated., Objectives: To investigate miRNA expression and function in MM and ZZ monocytes and identify miRNA(s) regulating the unfolded protein response., Methods: Peripheral blood monocytes were isolated from asymptomatic and symptomatic MM and ZZ individuals for miRNA expression profiling and pyrosequencing analysis. miRNA/gene and protein expression was measured with quantitative polymerase chain reaction and Western blotting. Overexpression and inhibition studies were performed with pre-miR or anti-miR, respectively. Luciferase reporter genes were used to elucidate direct miRNA-target interactions. Inflammatory cytokines were detected using the Meso Scale Discovery Plex assays., Measurements and Main Results: Forty-three miRNAs were differentially expressed, with miR-199a-5p most highly up-regulated in asymptomatic ZZ versus MM monocytes. miR-199a-2 promoter hypermethylation inhibits miR-199a-5p expression and was increased in symptomatic MM and ZZ monocytes compared with asymptomatic counterparts. GRP78, activating transcription factor 6, p50, and p65 were increased in symptomatic versus asymptomatic ZZ monocytes. Reciprocal down- or up-regulation of these markers was observed after miRNA modulation. Direct miR-199a-5p targeting of activating transcription factor 6, p50, and p65 by miR-199a-5p was demonstrated using luciferase reporter systems. Overexpression of miR-199a-5p also decreased other arms of the UPR and expression of cytokines that are not putative targets., Conclusions: miR-199a-5p is a key regulator of the unfolded protein response in AAT-deficient monocytes, and epigenetic silencing of its expression regulates this process in chronic obstructive pulmonary disease.
- Published
- 2014
- Full Text
- View/download PDF
24. MicroRNAs and liver cancer associated with iron overload: therapeutic targets unravelled.
- Author
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Greene CM, Varley RB, and Lawless MW
- Subjects
- Animals, Apoptosis, Carcinoma, Hepatocellular etiology, Carcinoma, Hepatocellular genetics, Carcinoma, Hepatocellular secondary, Carcinoma, Hepatocellular therapy, Disease Progression, Genetic Testing, Genetic Therapy, Humans, Iron Overload complications, Iron Overload genetics, Liver pathology, Liver Neoplasms etiology, Liver Neoplasms genetics, Liver Neoplasms pathology, Liver Neoplasms therapy, MicroRNAs therapeutic use, Neoplasm Invasiveness, Predictive Value of Tests, Carcinoma, Hepatocellular metabolism, Iron Overload metabolism, Liver metabolism, Liver Neoplasms metabolism, MicroRNAs metabolism
- Abstract
Primary liver cancer is a global disease that is on the increase. Hepatocellular carcinoma (HCC) accounts for most primary liver cancers and has a notably low survival rate, largely attributable to late diagnosis, resistance to treatment, tumour recurrence and metastasis. MicroRNAs (miRNAs/miRs) are regulatory RNAs that modulate protein synthesis. miRNAs are involved in several biological and pathological processes including the development and progression of HCC. Given the poor outcomes with current HCC treatments, miRNAs represent an important new target for therapeutic intervention. Several studies have demonstrated their role in HCC development and progression. While many risk factors underlie the development of HCC, one process commonly altered is iron homeostasis. Iron overload occurs in several liver diseases associated with the development of HCC including Hepatitis C infection and the importance of miRNAs in iron homeostasis and hepatic iron overload is well characterised. Aberrant miRNA expression in hepatic fibrosis and injury response have been reported, as have dysregulated miRNA expression patterns affecting cell cycle progression, evasion of apoptosis, invasion and metastasis. In 2009, miR-26a delivery was shown to prevent HCC progression, highlighting its therapeutic potential. Several studies have since investigated the clinical potential of other miRNAs with one drug, Miravirsen, currently in phase II clinical trials. miRNAs also have potential as biomarkers for the diagnosis of HCC and to evaluate treatment efficacy. Ongoing studies and clinical trials suggest miRNA-based treatments and diagnostic methods will have novel clinical applications for HCC in the coming years, yielding improved HCC survival rates and patient outcomes.
- Published
- 2013
- Full Text
- View/download PDF
25. Regulation of cystic fibrosis transmembrane conductance regulator by microRNA-145, -223, and -494 is altered in ΔF508 cystic fibrosis airway epithelium.
- Author
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Oglesby IK, Chotirmall SH, McElvaney NG, and Greene CM
- Subjects
- 3' Untranslated Regions, Adult, Base Sequence, Cell Line, Chlorides metabolism, Cystic Fibrosis metabolism, Cystic Fibrosis microbiology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Female, Gene Expression, Gene Expression Profiling, Gene Knockdown Techniques, Genotype, Humans, Intracellular Space metabolism, Lung metabolism, Lung microbiology, Male, MicroRNAs chemistry, MicroRNAs metabolism, Pseudomonas aeruginosa metabolism, Respiratory Mucosa microbiology, Respiratory Mucosa pathology, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Gene Expression Regulation, MicroRNAs genetics, Respiratory Mucosa metabolism
- Abstract
Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is altered in individuals with the ΔF508 CFTR mutation. We previously reported differential expression of microRNA (miRNA) in CF airway epithelium; however, the role of miRNA in regulation of CFTR expression here remains unexplored. In this study, we investigated the role of upregulated miRNAs in CFTR regulation in vivo in bronchial brushings from individuals homozygous or heterozygous for ΔF508 CFTR, validated our observations in vitro, and assessed the impact of defective chloride ion conductance, genotype, and colonization status on miRNA expression. miRNA target prediction was performed in silico, and expression of miRNA and target genes were measured by quantitative real-time PCR and/or Western blotting. Overexpression and inhibition studies were performed with pre-miRs or antimiRs, respectively, and a luciferase reporter gene was used to elucidate direct miRNA-target interactions. miR-145, miR-223, and miR-494 were upregulated in CF versus non-CF bronchial brushings and cell lines; in ΔF508 CFTR homozygotes versus heterozygotes; in subjects positive for P. aeruginosa; and in cells treated with a CFTR inhibitor or IL-1β. Reciprocal downregulation or upregulation of CFTR gene and/or protein expression was observed after miRNA manipulation and direct miRNA-target relationships demonstrated via a reporter system containing a wild type or mutated full-length CFTR 3' untranslated region. Increased expression of miR-145, miR-223, and miR-494 in vivo in bronchial epithelium of individuals carrying the ΔF508 CFTR mutation correlates with decreased CFTR expression. Defective CFTR function, Pseudomonas colonization, and inflammation may affect miRNA expression and contribute to the regulation of ΔF508 CFTR.
- Published
- 2013
- Full Text
- View/download PDF
26. Isolation and identification of cell-specific microRNAs targeting a messenger RNA using a biotinylated anti-sense oligonucleotide capture affinity technique.
- Author
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Hassan T, Smith SG, Gaughan K, Oglesby IK, O'Neill S, McElvaney NG, and Greene CM
- Subjects
- Binding Sites, Biotinylation, Cell Line, Enzyme-Linked Immunosorbent Assay, Humans, Interleukin-8 genetics, MicroRNAs metabolism, RNA Precursors metabolism, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction, Secretory Leukocyte Peptidase Inhibitor genetics, Transcriptome, alpha 1-Antitrypsin genetics, alpha 1-Antitrypsin metabolism, MicroRNAs isolation & purification, Oligodeoxyribonucleotides, Antisense, RNA, Messenger isolation & purification
- Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that regulate expression by translational repression or messenger RNA (mRNA) degradation. Although numerous bioinformatic prediction models exist to identify miRNA-mRNA interactions, experimental validation of bona fide interactions can be difficult and laborious. Few methods can comprehensively identify miRNAs that target a single mRNA. We have developed an experimental approach to search for miRNAs targeting any mRNA using a capture affinity assay involving a biotinylated DNA anti-sense oligonucleotide. This method identifies miRNAs targeting the full length of the mRNA. The method was tested using three separate mRNA targets: alpha-1 antitrypsin (AAT) mRNA, interleukin-8 mRNA and secretory leucoprotease inhibitor mRNA. AAT mRNA-specific and total miRNAs from three different cell lines (monocytic THP-1, bronchial epithelial 16HBE14o- and liver HepG2 cells) were profiled, and validation studies revealed that AAT mRNA-specific miRNAs functionally target the AAT mRNA in a cell-specific manner, providing the first evidence of innate miRNAs selectively targeting and modulating AAT mRNA expression. Interleukin-8 and secretory leucoprotease inhibitor mRNAs and their cognate miRNAs were also successfully captured using this approach. This is a simple and an efficient method to potentially identify miRNAs targeting sequences within the full length of a given mRNA transcript.
- Published
- 2013
- Full Text
- View/download PDF
27. microRNAs in asthma: potential therapeutic targets.
- Author
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Greene CM and Gaughan KP
- Subjects
- Anti-Asthmatic Agents therapeutic use, Asthma drug therapy, Bronchi pathology, Bronchi physiopathology, Cell Proliferation, Disease Management, Genetic Predisposition to Disease genetics, Humans, Polymorphism, Single Nucleotide genetics, Anti-Asthmatic Agents pharmacology, Asthma physiopathology, MicroRNAs drug effects, MicroRNAs physiology
- Abstract
Purpose of Review: Asthma is a global disease affecting millions of people. Current treatments are largely symptomatic and, although often effective, can be associated with various side effects. microRNAs (miRNAs/miRs) are regulatory RNAs that affect protein synthesis. They represent new therapeutic targets, and medicines that target specific miRNAs may have potential in the treatment of asthma., Recent Findings: There have been a number of studies in the field of miRNA that implicate specific miRNAs in the pathophysiology of asthma. For example, studies using mouse models have identified miRNAs that are altered in response to allergen challenge. Certain miRNAs that are involved in the regulation of interleukin-13 and the TH2 response, key components of the asthmatic response, have been shown to be amenable to modulation by premiRs and antimiRs. Other studies have identified miRNAs that are implicated in bronchial smooth muscle hyperresponsiveness and proliferation. Single-nucleotide polymorphisms in miRNA responsive elements within asthma susceptibility genes, and also in miRNAs themselves, can also contribute to the asthma phenotype., Summary: Developing miRNA-based medicines to treat the pulmonary manifestations of asthma could yield therapeutics with new properties that have the potential to treat both the inflammation and hyperresponsivesness associated with this disease.
- Published
- 2013
- Full Text
- View/download PDF
28. Targeting miRNA-based medicines to cystic fibrosis airway epithelial cells using nanotechnology.
- Author
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McKiernan PJ, Cunningham O, Greene CM, and Cryan SA
- Subjects
- Cell Line, Cystic Fibrosis genetics, Cystic Fibrosis pathology, Epithelial Cells, Humans, MicroRNAs chemistry, Nanocapsules ultrastructure, Particle Size, Cystic Fibrosis therapy, Genetic Therapy methods, MicroRNAs administration & dosage, MicroRNAs genetics, Nanocapsules administration & dosage, Nanocapsules chemistry, Respiratory Mucosa physiopathology
- Abstract
Cystic fibrosis (CF) is an inherited disorder characterized by chronic airway inflammation. microRNAs (miRNAs) are endogenous small RNAs which act on messenger (m) RNA at a post transcriptional level, and there is a growing understanding that altered expression of miRNA is involved in the CF phenotype. Modulation of miRNA by replacement using miRNA mimics (premiRs) presents a new therapeutic paradigm for CF, but effective and safe methods of delivery to the CF epithelium are limiting clinical translation. Herein, polymeric nanoparticles are investigated for delivery of miRNA mimics into CF airway epithelial cells, using miR-126 as a proof-of-concept premiR cargo to determine efficiency. Two polymers, polyethyleneimine (PEI) and chitosan, were used to prepare miRNA nanomedicines, characterized for their size, surface (zeta) potential, and RNA complexation efficiency, and screened for delivery and cytotoxicity in CFBE41o- (human F508del cystic fibrosis transmembrane conductance regulator bronchial epithelial) cells using a novel high content analysis method. RNA extraction was carried out 24 hours post transfection, and miR-126 and TOM1 (target of Myb1) expression (a validated miR-126 target) was assessed. Manufacture was optimized to produce small nanoparticles that effectively complexed miRNA. Using high content analysis, PEI-based nanoparticles were more effective than chitosan-based nanoparticles in facilitating uptake of miRNA into CFBE41o- cells and this was confirmed in miR-126 assays. PEI-premiR-126 nanoparticles at low nitrogen/phosphate (N/P) ratios resulted in significant knockdown of TOM1 in CFBE41o- cells, with the most significant reduction of 66% in TOM1 expression elicited at an N/P ratio of 1:1 while chitosan-based miR-126 nanomedicines failed to facilitate statistically significant knockdown of TOM1 and both nanoparticles appeared relatively nontoxic. miRNA nanomedicine uptake can be qualitatively and quantitatively assessed rapidly by high content analysis and is highly polymer-dependent but, interestingly, there is not a direct correlation between the levels of miRNA uptake and the downstream gene knockdown. Polymeric nanoparticles can deliver premiRs effectively to CFBEs in order to modulate gene expression but must be tailored specifically for miRNA delivery.
- Published
- 2013
- Full Text
- View/download PDF
29. Therapeutic modulation of miRNA for the treatment of proinflammatory lung diseases.
- Author
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Hassan T, McKiernan PJ, McElvaney NG, Cryan SA, and Greene CM
- Subjects
- Gene Targeting methods, Humans, Immunity, Innate drug effects, Immunity, Innate genetics, Inflammation physiopathology, Lung Diseases physiopathology, MicroRNAs antagonists & inhibitors, MicroRNAs genetics, Gene Expression Regulation drug effects, Inflammation therapy, Lung Diseases therapy, MicroRNAs metabolism
- Abstract
miRNAs are short, nonprotein coding RNAs that regulate target gene expression principally by causing translational repression and/or mRNA degradation. miRNAs are involved in most mammalian biological processes and have pivotal roles in controlling the expression of factors involved in basal and stimulus-induced signaling pathways. Considering their central role in the regulation of gene expression, miRNAs represent therapeutic drug targets. Here we describe how miRNAs are involved in the regulation of aspects of innate immunity and inflammation, what happens when this goes awry, such as in the chronic inflammatory lung diseases cystic fibrosis and asthma, and discuss the current state-of-the-art miRNA-targeted therapeutics.
- Published
- 2012
- Full Text
- View/download PDF
30. MicroRNAs in inflammatory lung disease--master regulators or target practice?
- Author
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Oglesby IK, McElvaney NG, and Greene CM
- Subjects
- Humans, Gene Expression Regulation, Gene Targeting, Inflammation physiopathology, Lung metabolism, Lung Diseases physiopathology, MicroRNAs genetics, MicroRNAs metabolism
- Abstract
MicroRNAs (miRNAs) have emerged as a class of regulatory RNAs with immense significance in numerous biological processes. When aberrantly expressed miRNAs have been shown to play a role in the pathogenesis of several disease states. Extensive research has explored miRNA involvement in the development and fate of immune cells and in both the innate and adaptive immune responses whereby strong evidence links miRNA expression to signalling pathways and receptors with critical roles in the inflammatory response such as NF-κB and the toll-like receptors, respectively. Recent studies have revealed that unique miRNA expression profiles exist in inflammatory lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis and lung cancer. Evaluation of the global expression of miRNAs provides a unique opportunity to identify important target gene sets regulating susceptibility and response to infection and treatment, and control of inflammation in chronic airway disorders. Over 800 human miRNAs have been discovered to date, however the biological function of the majority remains to be uncovered. Understanding the role that miRNAs play in the modulation of gene expression leading to sustained chronic pulmonary inflammation is important for the development of new therapies which focus on prevention of disease progression rather than symptom relief. Here we discuss the current understanding of miRNA involvement in innate immunity, specifically in LPS/TLR4 signalling and in the progression of the chronic inflammatory lung diseases cystic fibrosis, COPD and asthma. miRNA in lung cancer and IPF are also reviewed.
- Published
- 2010
- Full Text
- View/download PDF
31. miR-126 is downregulated in cystic fibrosis airway epithelial cells and regulates TOM1 expression.
- Author
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Oglesby IK, Bray IM, Chotirmall SH, Stallings RL, O'Neill SJ, McElvaney NG, and Greene CM
- Subjects
- Bronchi immunology, Bronchi metabolism, Bronchi pathology, Cell Line, Cell Line, Tumor, Cells, Cultured, Cystic Fibrosis genetics, Down-Regulation genetics, Female, Humans, Immunity, Innate genetics, Intracellular Signaling Peptides and Proteins, Male, Middle Aged, Proteins genetics, Respiratory Mucosa pathology, U937 Cells, Young Adult, Cystic Fibrosis immunology, Down-Regulation immunology, MicroRNAs antagonists & inhibitors, MicroRNAs physiology, Proteins metabolism, Respiratory Mucosa immunology, Respiratory Mucosa metabolism
- Abstract
Cystic fibrosis (CF) is one of the most common lethal genetic diseases in which the role of microRNAs has yet to be explored. Predicted to be regulated by miR-126, TOM1 (target of Myb1) has been shown to interact with Toll-interacting protein, forming a complex to regulate endosomal trafficking of ubiquitinated proteins. TOM1 has also been proposed as a negative regulator of IL-1beta and TNF-alpha-induced signaling pathways. MiR-126 is highly expressed in the lung, and we now show for the first time differential expression of miR-126 in CF versus non-CF airway epithelial cells both in vitro and in vivo. MiR-126 downregulation in CF bronchial epithelial cells correlated with a significant upregulation of TOM1 mRNA, both in vitro and in vivo when compared with their non-CF counterparts. Introduction of synthetic pre-miR-126 inhibited luciferase activity in a reporter system containing the full length 3'-untranslated region of TOM1 and resulted in decreased TOM1 protein production in CF bronchial epithelial cells. Following stimulation with LPS or IL-1beta, overexpression of TOM1 was found to downregulate NF-kappaB luciferase activity. Conversely, TOM1 knockdown resulted in a significant increase in NF-kappaB regulated IL-8 secretion. These data show that miR-126 is differentially regulated in CF versus non-CF airway epithelial cells and that TOM1 is a miR-126 target that may have an important role in regulating innate immune responses in the CF lung. To our knowledge, this study is the first to report of a role for TOM1 in the TLR2/4 signaling pathways and the first to describe microRNA involvement in CF.
- Published
- 2010
- Full Text
- View/download PDF
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