miR-224-5p and miR-545-5p Levels Relate to Exacerbations and Lung Function in a Pilot Study of X-Linked MicroRNA Expression in Cystic Fibrosis Monocytes.

Altered microRNA expression patterns in bronchial brushings from people with versus without cystic fibrosis (CF) relate to functional changes and disease pathophysiology. The expression of microRNAs encoded on the X chromosome is also altered in peripheral blood monocytes of p. Phe508del homozygous versus non-CF individuals. Here we investigate whether levels of the top seven X-linked microRNAs (miR-224-5p, miR-452-5p, miR-450b-5p, miR-542-3p, miR-450a-5p, miR-424-5p, and miR-545-5p) that are significantly increased over 1.5 fold in CF versus non-CF monocytes correlate with lung function. CD14+ monocytes were isolated from males and females with (n = 12) and without cystic fibrosis (n = 12) and examined for the expression of X-linked microRNAs by qRT-PCR array. MicroRNA target mRNA levels were quantified using qRT-PCR. Clinical correlations with lung function data were analysed in the CF cohort. Increasing levels of miR-545-5p correlated moderately with FEV1% predicted (r = -0.4553, p > 0.05) and strongly with exacerbation rate (r = 0.5858, p = 0.0483). miR-224-5p levels were significantly higher in the severe (FEV1 <40%) versus mild (FEV1 ≥80%, p = 0.0377) or moderate (FEV1 40-79%, p = 0.0350) groups. MiR-224-5p expression inversely correlated with lung function (FEV1%: r = -0.5944, p = 0.0457) and positively correlated with exacerbation rates (r = 0.6139, p = 0.0370). These data show that peripheral blood monocyte miR-545-5p and miR-224-5p levels correlate with exacerbation rate, whilst miR-224-5p levels also correlate with lung function in cystic fibrosis.

The Estrogen-Induced miR-19 Downregulates Secretory Leucoprotease Inhibitor Expression in Monocytes.

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 17ß-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.

Knockdown of Gene Expression in Macrophages by microRNA Mimic-Containing Poly (Lactic-co-glycolic Acid) Microparticles.

Background: microRNA (miRNA) regulate target gene expression through translational repression and/or mRNA degradation and are involved in the regulation of inflammation. Macrophages are key inflammatory cells that are important in chronic inflammatory lung diseases such as cystic fibrosis (CF). Macrophage-expressed miRNA represent therapeutic drug targets, yet delivery of nucleic acids to macrophages has proved challenging. Methods: miRNAs were encapsulated in poly (lactic-co-glycolic acid) (PLGA)-based microparticles using double emulsion solvent evaporation and characterised for physicochemical features. Phorbol myristic acetate (PMA)-differentiated U937 macrophages were transfected with empty PLGA microparticles or those encapsulating a premiR-19b-3p or scrambled control miRNA mimic. miRNA internalisation and knockdown of a miR-19b-3p target gene, secretory leucoprotease inhibitor (SLPI), were determined by qRT-PCR. Results: Microparticle formulations were consistently found to be 2⁻3µm and all had a negative ζ potential (-5 mV to -14 mV). Encapsulation efficiency of premiR-19b-3p was 37.6 ± 13.4%. Levels of mature miR-19b-3p were higher in macrophages after delivery of premiR-19b-3p microparticles compared to empty or scrambled control miRNA-containing microparticles. Significant SLPI knockdown was achieved 72 hours post-delivery of premiR-19b-3p microparticles compared to controls. Conclusions: miRNA-encapsulating PLGA microparticles offer a new treatment paradigm for delivery to macrophages that could potentially be administered to CF lungs via inhalation.

Expression of X-linked Toll-like receptor 4 signaling genes in female vs. male neonates.

BACKGROUND: Male neonates display poorer disease prognosis and outcomes compared with females. Immune genes which exhibit higher expression in umbilical cord blood (UCB) of females may contribute to the female immune advantage during infection and inflammation. The aim of this study was to quantify expression of Toll-like receptor (TLR) 4 signaling genes encoded on the X-chromosome in UCB from term female vs. male neonates. METHODS: UCB samples were collected from term neonates (n = 26) born by elective Caesarean section and whole blood was collected from adults (n = 20). Leukocyte RNA was isolated and used in quantitative PCR reactions for IκB kinase γ (IKKγ), Bruton's tyrosine kinase (BTK), and IL-1 receptor associated kinase (IRAK)1. IRAK1 protein was analyzed by Western blot and confocal microscopy. RESULTS: In neonates there was no significant difference in the relative expression of IKKγ or BTK mRNA between genders. IRAK1 gene and protein expression was significantly higher in female vs. male UCB, with increased cytosolic IRAK1 expression also evident in female UCB mononuclear cells. Adults had higher expression of all three genes compared with neonates. CONCLUSION: Increased expression of IRAK1 could be responsible, in part, for sex-specific responses to infection and subsequent immune advantage in female neonates.

MiRNA Expression in Cystic Fibrosis Bronchial Epithelial Cells.

Bronchial epithelial cells represent an invaluable tool to elucidate molecular signaling regulation in cystic fibrosis (CF). CF is a lethal genetic condition characterized by chronic inflammation in which bronchial epithelial cells play a pivotal role. Here we describe their use in analysis of microRNA (miRNA) and their target genes following a two-step RT-PCR miRNA profiling method in bronchial cell specimens from CF and control individuals where 667 human miRNA were examined. We also describe an approach to experimental modulation of these miRNA in vitro.

High-throughput profiling for discovery of non-coding RNA biomarkers of lung disease.

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.

MicroRNA Dysregulation in Cystic Fibrosis.

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.

Non-coding RNA as lung disease biomarkers.

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.

Long noncoding RNA are aberrantly expressed in vivo in the cystic fibrosis bronchial epithelium.

Long non-coding RNAs (lncRNAs) have emerged recently as key regulatory molecules with diverse roles at almost every level of the regulation of gene expression. The roles of these RNAs in the pathogenesis of cystic fibrosis (CF); a lethal multisystem, autosomal recessive disorder have yet to be explored. Our aim was to examine the expression profile of lncRNA, in the airway epithelium of people with CF. We examined the expression of 30,586 lncRNAs by microarray (Human LncRNA Array v3.0, Arraystar, Inc.), in vivo in bronchial cells isolated from endobronchial brushings obtained from CF and non-CF individuals. In total, we identified 1,063 lncRNAs with differential expression between CF and non-CF individuals (fold change ≥3, p≤0.001). The microarray also contained probes for ∼26,109 protein coding transcripts, of which 720 were differentially expressed between CF and non-CF brush samples (fold change ≥3, p≤0.001). Confirmation of a selection of differentially expressed coding mRNA and lncRNA transcripts such as XIST and TLR8 was achieved using qRT-PCR. Gene ontology bioinformatics analysis highlighted that many processes over-represented in the CF bronchial epithelium are related to inflammation. These data show a significantly altered lncRNA and mRNA expression profile in CF bronchial cells in vivo. Dysregulation of some of these lncRNAs may play important roles in the chronic infection and inflammation that exists in the lungs of people with CF.

Targeting miRNA-based medicines to cystic fibrosis airway epithelial cells using nanotechnology.

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.

Therapeutic modulation of miRNA for the treatment of proinflammatory lung diseases.

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.

SLPI and inflammatory lung disease in females.

During the course of certain inflammatory lung diseases, SLPI (secretory leucoprotease inhibitor) plays a number of important roles. As a serine antiprotease it functions to protect the airways from proteolytic damage due to neutrophil and other immune cell-derived serine proteases. With respect to infection it has known antimicrobial and anti-viral properties that are likely to contribute to host defence. Another of its properties is the ability to control inflammation within the lung where it can interfere with the transcriptional induction of pro-inflammatory gene expression induced by NF-κB (nuclear factor κB). Thus, factors that regulate the expression of SLPI in the airways can impact on disease severity and outcome. Gender represents once such idiosyncratic factor. In females with CF (cystic fibrosis), it is now thought that circulating oestrogen contributes, in part, to the observed gender gap whereby females have worse disease and poorer prognosis than males. Conversely, in asthma, sufferers who are females have more frequent exacerbations at times of low-circulating oestrogen. In the present paper, we discuss how SLPI participates in these events and speculate on whether regulatory mechanisms such as post-transcriptional modulation by miRNAs (microRNAs) are important in the control of SLPI expression in inflammatory lung disease.