Paul J. McKiernan

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.

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.

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.

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.

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.

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.

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

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.

The Biology of Long Non-Coding RNA

The majority of human transcribed loci do not encode protein. Long non-coding RNAs (lncRNAs) are an emerging class of non-coding RNA that are implicated in the regulation of gene expression at almost every level. Although their mechanisms of action are relatively unknown, general mechanistic themes are emerging. They have the ability to affect gene expression through a spectrum of interactions with RNA, proteins and possibly DNA. With their capacity to act both in cis and in trans, they can guide epigenetic-modifier complexes or transcription factors to particular genomic sites. These lncRNAs have been implicated in a variety of cellular processes, including chromatin modification, genomic imprinting and cell cycle regulation. This chapter examines their biology, conservation, function and roles in disease as well as various approaches to their characterisation.

152 Estrogen-regulated MicroRNAs control the expression of secretory leukoprotease inhibitor in monocytes

Apoptosis is a physiological process essential for homeostasis of epithelial organisation and function. CF lung disease is characterised by chronic infection and inflammation and previous work suggests that apoptosis is dysfunctional in the CF airways with conflicting results. In addition, controversy exists regarding how CFTR misfolding contributes to apoptosis. In this study, we evaluated the relationship between CFTR mutation and apoptosis in DF508-CFTR CF airway epithelial cells. Basal activity of the executioner caspase, caspase-3, was significantly increased in CF tracheal and bronchial epithelial cell lines and primary bronchial epithelial cells compared to non-CF controls. In addition, activity of the upstream initiator caspase, caspase-8, was significantly increased in CF epithelial cells compared to controls, suggesting involvement of extrinsic apoptosis signalling, which is mediated by the activation of death receptors, such as Fas (CD95). Increased levels of Fas were observed in CF epithelial cells, and neutralization of Fas significantly inhibited caspase-3 activity in CF epithelial cells compared to untreated cells. Furthermore, activation of Fas significantly increased caspase-3 activity and apoptosis in CF epithelial cells compared to control cells. Overall, these results suggest that CF airway epithelial cells are more sensitive to apoptosis via increased levels of Fas and subsequent activation of the Fas death receptor pathway.