Non-coding RNAs (ncRNAs) are an abundant class of RNAs that include small ncRNAs, long non-coding RNAs (lncRNA) and pseudogenes. The human ncRNA atlas includes thousands of these specialised RNA molecules that are further subcategorised based on their size or function. Two of the more well-known and widely studied ncRNA species are microRNAs (miRNAs) and lncRNAs. These are regulatory RNAs and their altered expression has been implicated in the pathogenesis of a variety of human diseases. Failure to express a functional cystic fibrosis (CF) transmembrane receptor (CFTR) chloride ion channel in epithelial cells underpins CF. Secondary to the CFTR defect, it is known that other pathways can be altered and these may contribute to the pathophysiology of CF lung disease in particular. For example, quantitative alterations in expression of some ncRNAs are associated with CF. In recent years, there has been a series of published studies exploring ncRNA expression and function in CF. The majority have focussed principally on miRNAs, with just a handful of reports to date on lncRNAs. The present study reviews what is currently known about ncRNA expression and function in CF, and discusses the possibility of applying this knowledge to the clinical management of CF in the near future.

Introduction

Cystic fibrosis (CF) is a single gene disorder. Although it is a multisystem disease, its major pathology is chronic airway inflammation that ultimately leads to a progressive decline in lung function. The primary cause of CF is a defect in the cystic fibrosis transmembrane receptor (CFTR) gene; chronic inflammation and infection are secondary events that contribute significantly to CF and affect the lung disease in particular. In addition to what is known regarding the effects of dysfunctional CFTR protein in the lung, there is emerging evidence for important roles for non-coding RNAs (ncRNAs) in the pathophysiology of CF. Herein, we review the current knowledge regarding ncRNA expression and function in CF and explain the properties and known roles of ncRNAs.

Cystic fibrosis

CF is an autosomal recessive genetic disease caused by mutations in CFTR. This gene encodes an important chloride ion channel expressed on the surface of epithelial cells in the lung and other organs. There are over 1800 different mutations that have been identified, and these are classed into groups according to their implications for the CFTR protein, e.g. defective synthesis, processing, trafficking, channel function and stability [1]. Different mutations result in varying degrees of severity of the disease, depending on the overall capacity of CFTR to function properly at the apical membrane. The most common mutation worldwide is p.Phe508del, which is a deletion of a phenylalanine residue that causes the protein to be degraded in the endoplasmic reticulum (ER) of the cell. Very little CFTR reaches the cell surface and that which does is dysfunctional, therefore a patient with two copies of this allele would experience a severe form of the disease.

Although other organs in the body experience difficulties as a result of defective CFTR, the severity of the disease is most apparent in the lung. The consequence of inadequate CFTR function in the CF airway lumen is an imbalance between CFTR-mediated Cl secretion and epithelial sodium channel (ENaC)-mediated Na+ absorption, causing a depletion in airway surface liquid volume and an excessively dehydrated and viscous mucus lining the airway. The lung, therefore, becomes an ideal environment for colonisation by pathogenic microorganisms such as Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia and Aspergillus fumigatus [2]. The inflammatory response of the CF lung to infections such as these is known to be dysregulated from numerous perspectives, with the hallmark characteristics of the disease recognised as being: hyper-reactive pattern recognition receptor signalling (e.g. Toll-like receptors/TLRs), higher levels of pro-inflammatory cytokines (e.g. IL-8), excessive neutrophil recruitment and an excessive burden of protease activity (e.g. neutrophil elastase) [3]. The repeated exacerbations of infection and inflammation progressively destroy healthy lung tissue over the course of the patient's life, eventually resulting in death due to respiratory failure.

There is no known cure for CF, and hence current treatments for patients are primarily centred around management of the symptoms created as a consequence of impaired CFTR. These include: physical therapy to aid mucus clearance (in combination with mucolytic pharmacological agents), antibiotics against airway infections and anti-inflammatory treatments such as macrolides [4]. The development of better antibiotics is widely regarded as the main factor resulting in the increased life expectancy for CF patients in the past decade [5]. However, with the constant threat of antibiotic resistance looming on the horizon, there is an urgent need for alternative therapy options. Until recently, many attempts at correcting CFTR ion channel function had been made, but none had shown hope of achieving sufficient efficacy. New approaches are now showing promising signs of improving patient lung function, e.g. the use of the CFTR potentiator, ivacaftor, in combination with the CFTR corrector, lumacaftor (both Vertex Pharmaceuticals Incorporated, Cambridge, MA) [6]. In addition, many other research groups are continuously identifying novel pathways which are altered in CF and could be exploited as possible therapeutic strategies, one example of which is the role of ncRNA in the disease pathogenesis.

Description of ncRNA classes

The human genome contains ∼3 billion base pairs and is structured to form a complex and packaged architecture, called chromatin, which resides inside the nucleus of eukaryotic cells. The draft sequence of the human genome was first published in 2001 [7,8], and 3 years later the ‘gold standard' sequence appeared in Nature suggesting the presence of 20 000–25 000 protein-coding genes [9].

The GENCODE Consortium aimed to annotate all genes in the human genome with high accuracy, using a combination of manual annotation, computational analyses and experimental validation [10]. The current human GENCODE Release (version 27) contains 19 836 protein-coding genes (representing ∼2% of the human genome), in juxtaposition with thousands of ncRNAs, including pseudogenes (n = 14 694), long non-coding RNAs (lncRNAs) (n = 15 778) and small ncRNAs (n = 7569), as represented in Figure 1A. NcRNAs can be classified by length (small 18–200 nt; long >200 nt) or by function [housekeeping ncRNAs, such as rRNAs and tRNAs; regulatory transcripts, such as microRNAs (miRNAs), piwi-interacting RNA, lncRNA]. Since the role of housekeeping ncRNAs is very well characterised, research in the last two decades has been focussing on regulatory ncRNAs.

Current annotation for the human genome according to the latest GENCODE releases, version 27 (January 2017 freeze, GRCh38 — Ensembl 90, 91).

Figure 1.
Current annotation for the human genome according to the latest GENCODE releases, version 27 (January 2017 freeze, GRCh38 — Ensembl 90, 91).

(A) Genomic loci are divided in four different categories — protein coding, pseudogenes and long and small non-coding RNA genes. Abundance of the main classes of (B) pseudogenes, (C) long and (D) small ncRNA loci according to GENCODE27. Abbreviations: lincRNA, long intervening (or intergenic) non-coding RNA; NAT, natural antisense transcript; miRNA, microRNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; piRNA, piwi-interacting RNA; ‘?' indicates that the number is not based on GENCODE27 but on an external database.

Figure 1.
Current annotation for the human genome according to the latest GENCODE releases, version 27 (January 2017 freeze, GRCh38 — Ensembl 90, 91).

(A) Genomic loci are divided in four different categories — protein coding, pseudogenes and long and small non-coding RNA genes. Abundance of the main classes of (B) pseudogenes, (C) long and (D) small ncRNA loci according to GENCODE27. Abbreviations: lincRNA, long intervening (or intergenic) non-coding RNA; NAT, natural antisense transcript; miRNA, microRNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; piRNA, piwi-interacting RNA; ‘?' indicates that the number is not based on GENCODE27 but on an external database.

Pseudogenes

Originally discovered in Xenopus laevis in 1977 [11], pseudogenes are DNA sequences that somehow resemble functional genes, but they have lost their ability to be translated into proteins and have been subsequently considered as ‘genomic fossils' valuable mostly for understanding evolution of genes and genomes [12].

They can be categorised according to their formation mechanism into three main groups, represented in Figure 1B [13]: (i) processed pseudogenes (n = 10 704 in GENCODE27) derive from retrotransposition processes where mRNAs undergo reverse transcription into cDNA and reinsertion into the genome in a new location; (ii) unprocessed pseudogenes (n = 3469) are formed through duplication of protein-coding genes followed by mutations in the duplicated sequence, thereby leading to loss of function in terms of protein production; (iii) unitary pseudogenes (n = 206) are single copy parent genes that become non-functional after accumulation of spontaneous mutations, therefore they do not have a genomic counterpart encoding a fully functional protein.

Although they have been labelled as ‘junk' DNA for a long time, recent reports have described that pseudogenes can act as miRNA decoys in competition with mRNAs [14,15] and as a source of endogenous siRNAs to silence protein-coding transcripts [16]. Recent discoveries about their altered expression and function provide clues for their significant role in the pathogenesis of cancer [17] and neurodegenerative diseases [18]. Although their potential biological roles have not been addressed in CF, mutations within CFTR pseudogenes were reported to affect the accuracy of CF carrier testing [19,20].

Long non-coding RNAs

LncRNAs are defined as RNA molecules longer than 200 nt that do not encode any protein product. One of the first reported and most important lncRNAs is the X inactive-specific transcript (XIST), described in 1996 as a master regulator of the X-chromosome inactivation process [21]. Researchers have shown in the past 10 years that lncRNAs have a very broad repertoire of functions in chromatin modification and in transcriptional, post-transcriptional and translational regulation [22]. Due to their significant effect on these biological processes, they are reported to be heavily involved in several pathologies including cancer [23], cardiovascular diseases [24] and diabetes [25]. Their expression and function in CF have recently begun to be explored as discussed later in this review.

LncRNAs can be loosely classified based on their genomic position with respect to protein-coding genes, as shown in Figure 1C [26]: (i) long intervening (or intergenic) ncRNAs (lincRNAs, n = 7499), including XIST, are found between protein-coding genes, without overlap with protein-coding genes or other ncRNAs genes; (ii) natural antisense transcripts (NATs, n = 5521) are transcribed from the opposite strand of other annotated transcripts, and thus share (usually partial) sequence complementarity; (iii) intronic lncRNAs (n = 905) are found within intronic regions of other genes, although it is not clear yet whether this class of lncRNAs arises from mRNA splicing or is actually independently transcribed.

Although not clearly annotated in GENCODE as a separate group (yet), the covalently closed RNA loops called ‘circular RNAs' (circRNAs) can be considered as lncRNAs. The details of their biogenesis are still unclear; however, they are thought to be generated by back-splicing circularisation catalysed by the spliceosome machinery, and to be neither capped nor polyadenylated [27]. The possibility that RNAs could assume a circular shape was first proposed in the late 1970s following imaging of eukaryotic cells by electron microscopy [28]. Although some circRNAs were identified in the following years [2931], they were mainly considered as ‘splicing noise' until 2012 when deep sequencing data from normal and malignant human cells found thousands of RNA transcripts in which exons were arranged in a non-canonical order [32]. This observation was further corroborated in 2013 when Jeck et al. showed that fibroblast RNAs treated with RNAse R contained more than 25 000 circRNAs, with a higher stability than the linear counterpart [33]. Eventually, two back-to-back articles in Nature officially acknowledged circRNAs as a class of ncRNAs [34,35].

Their biological functions have not been explored fully yet, but recent insights proposed that circRNAs could play a role in ‘sponging' miRNAs, sequestering ribonucleoproteins and regulating transcription, ultimately affecting the pathogenesis of cancer, cardiovascular diseases and neurological disorders [36]. Ongoing research in this group is focussed on the contribution of circRNAs to the pathophysiology of CF.

Small ncRNAs

Small ncRNAs are defined as molecules shorter than 200 nt that do not encode any protein product. There are many species of small ncRNAs and the most important are represented in Figure 1D and described as follows:

  • miRNAs (n = 1881): 20–25 nt in length, they were first identified in the early 1990s in C. elegans [37], and have captured researchers' attention since then due to their high conservation and great regulatory potential. Canonical miRNA biogenesis in animals undergoes a stepwise process [38] that leads to the generation of mature single-stranded miRNAs involved in the post-transcriptional regulation of ∼60% of protein-coding genes [39]. miRNAs negatively regulate gene expression by base pairing to miRNA recognition elements located usually within the 3′untranslated region (UTR) of the target mRNAs, thereby causing translational repression and/or mRNA degradation. Aberrations in miRNA genetic sequences, biogenesis and function have been associated with almost all the physiological and pathological processes investigated to date, including aging [40], cancer [41] and autoimmune diseases [42]. Their role in CF has been explored by several research groups, as outlined below.

  • Small nuclear RNAs (snRNAs) (n = 1900): this class comprises a small group of highly abundant and non-polyadenylated transcripts located in the nucleoplasm with an average length of 150 nt. The snRNAs can be divided in two categories [43]: (i) Sm-class RNAs (including U1, U2, U4, U4atac, U5, U7, U11 and U12) are transcribed by a specialised form of RNA polymerase II and characterised by a binding site for a group of seven Sm proteins; (ii) Lsm-class RNAs (including U6 and U6atac) are transcribed by RNA polymerase III and contain a binding site for the Lsm proteins. When associated with these specific proteins to become snRNPs, they form the core of the spliceosome and catalyse the removal of introns from pre-mRNAs, with the exception of U7 snRNP, which is involved in the 3′ processing of histone mRNAs [44]. Interestingly, spinal muscular atrophy is an autosomal recessive disease caused by mutations in the SMN1 gene, encoding one of the Sm proteins involved in the formation of snRNPs [45], and overexpression of U1 snRNA is associated with the development of Alzheimer's disease [46]. Although their role has not been explored in CF yet, a type of U1 snRNA directed specifically to CFTR exon 12 has been proposed as a potential therapeutic strategy to correct splicing mutations [47].

  • Small nucleolar RNAs (snoRNAs) (n = 943): ranging from 60 to 300 nt, they are grouped in two families (i.e. the C/D and the H/ACA RNAs) and are located in the nucleolar areas of the cell [43]. Although mainly involved in rRNA and tRNA chemical modifications, they have been recently shown to play a role in mRNA 3′ processing [48] and cholesterol homeostasis [49]. Aberrant expression or genetic alteration of snoRNA genes has been associated with various types of cancer and X-linked dyskeratosis congenita [50]. No implications of this class of ncRNAs in CF are known to date.

  • Piwi-interacting RNAs (piRNAs) (n = 6229?): although not individually annotated in GENCODE, these ncRNAs are 24–30 nt long and mainly expressed in the germline. PiRNAs are considered the guardians of the germline genome since by binding to Piwi-class proteins they are responsible for the silencing of transposable elements, thereby ensuring genomic stability during meiosis [51]. They are involved in several other mechanisms including epigenetic regulation, gene and protein regulation, and genome rearrangement [52]. PiRNA genes are organised in genomic clusters which range between 1 and 100 kb in size and encode between 10 and 4500 piRNAs, although a smaller fraction appears to map to a handful of protein-coding genes [53]. According to a recently developed database [54], the number of mature piRNAs is over 6000; they are reported to have a potential role in somatic cells too [55], and their aberrant expression and function are associated with human pathologies such as cancer [52] and Alzheimer's disease [56]. No studies on piRNAs in CF have been published to date.

miRNA studies in cystic fibrosis

Innate immunity/inflammation

The reduction in CFTR ion channel function in the CF airway epithelium leads to a dehydrated lumen with impaired mucociliary clearance, driving chronic pulmonary inflammation. Since airway epithelial cells show high expression of TLRs, they act as important mediators of the innate immune response. We were the first group to report that miRNA expression is altered in CF and that this could affect the inflammatory response in bronchial epithelial cells. Our expression profiling study compared bronchial brushings from five CF patients versus five non-CF controls, and the results identified 92 differentially expressed miRNAs (56 down-regulated and 36 up-regulated) [57]. miR-126 was selected and further experiments validated its significant down-regulation in both CF bronchial brushings relative to healthy controls, and in the CF bronchial epithelial cell line, CFBE41o, relative to the healthy bronchial epithelial cell line, 16HBE14o. This down-regulation correlated with a significant up-regulation of its target mRNA, TOM1 [57]. TOM1 (target of Myb1) is involved in the endosomal trafficking of ubiquitinated proteins and also functions as a negative regulator of the TLR2, TLR4 and IL-1R1 signalling pathways, therefore decreased levels of miR-126 have important implications for innate immune responses in the CF airway.

From a panel of differentially expressed miRNAs identified in a comparison between the CF and non-CF bronchial epithelial cell lines IB3-1 and IB3-1/S9, respectively, miR-155 emerged as a candidate for further investigation [58]. Levels of miR-155 were elevated in CF cells (in vitro and in vivo), and its up-regulation was shown to cause decreased expression of SHIP1 (Src homology-2 domain-containing inositol 5-phosphatase 1), thereby allowing increased PI3K/Akt (phosphatidylinositol 3-kinase) signalling and stabilisation of the IL-8 mRNA transcript [58]. In an attempt to identify mechanisms responsible for increased miR-155 expression in CF cells, a subsequent study by the same group demonstrated that the mRNA-destabilising protein TTP (tristetraprolin, found at unusually low levels in the CF lung) is a negative regulator of miR-155 biogenesis, potentially through the induction of miR-1 [59].

CF lung disease is characterised by a neutrophil-dominated airway inflammatory response. The major neutrophil chemokine IL-8 is present at elevated levels in the CF lung and thereby contributes to neutrophil recruitment, as well as triggering other pro-inflammatory signals. Strategies to reduce IL-8 expression hold therapeutic potential for CF patients. In addition to the effects of miR-155 on IL-8 expression, both miR-93 and miR-17 have been demonstrated to directly target the IL-8 transcript. miR-17 was found to be down-regulated in CF bronchial brushings, and both miR-17 and miR-93 were decreased during Pseudomonas-induced inflammation in bronchial epithelial cell lines, thereby enhancing IL-8 production [60,61].

Other cell types involved in CF pathogenesis include macrophages, which are also observed to be increased in the lungs of CF patients and in mouse models of the disease, again with implications for driving innate immune signalling. Zhang et al. [62] reported that human and murine CF macrophages express reduced levels of CAV1 (caveolin 1), due to post-transcriptional regulation by increased miR-199a-5p. CAV1 normally functions as a negative regulator of the TLR4 pathway and hence this finding may represent one mechanism by which TLR4 signalling remains ‘switched on' in the CF lung environment. TGF-β1 (transforming growth factor beta) signalling can also function to negatively regulate the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells)/IL-8 axis, and interestingly the TGF-β signal transducers SMAD3 and SMAD4 were highlighted in separate studies as being significantly decreased in CF cells [63,64]. Levels of these mRNA transcripts inversely correlated with increased levels of their validated targets, miR-145 (SMAD3) and miR-224-5p (SMAD4), highlighting yet another mechanism which may contribute to hyperactive pro-inflammatory signalling in the CF lung [63,64].

The dysregulated inflammatory response encountered in the CF lung is also characterised by a high burden of proteases released from activated immune cells. Neutrophil elastase has long been recognised as one of the most potent and important proteases in this regard; however, recent evidence is also building around the destructive and pro-inflammatory role of cathepsin S (CTSS) in CF lung pathology. Weldon et al. [65] found that elevated CTSS in CF bronchoalveolar lavage fluid correlated with poorer lung function. miR-31 was shown to regulate CTSS expression via targeting of the transcription factor IRF-1 (interferon regulatory factor 1), and miR-31 levels were significantly lower in bronchial brushings from CF versus non-CF patients [65].

Bacterial clearance

In addition to altered inflammatory signalling, CF macrophages are also defective in intracellular bacterial killing. A recent report by Pierdomenico et al. [66] revealed an interesting role for miR-181b in the impairment of CF macrophage function. miR-181b was elevated in the CFBE41o cell line and in primary monocyte-derived macrophages from CF patients. This was matched with lower expression of its validated target, ALX/FPR2 (lipoxin A4 receptor/formyl-peptide receptor type 2), which is a cell surface receptor recognised by the pro-resolution molecule lipoxin A4. Transfection of CF macrophages with a miR-181b inhibitor resulted in almost full restoration of lipoxin A4-induced phagocytotic ability, when compared with non-CF macrophages [66].

In contrast with the reduction in miR-17 in CF bronchial brushings, miR-17 was found to be up-regulated in primary CF macrophages of human and murine origin, and loss of the Mirc1/miR-17-92 cluster from these cells resulted in increased levels of the miR-17 validated targets ATG7 and ATG16L1 [67]. Inhibition of miR-17 in the lungs of CF mice increased the expression of these autophagy molecules and enhanced defective autophagy activity as measured by improved clearance of bacterial infection [67]. The enhancement of autophagy also led to improved CFTR function [67]. Interestingly, Mirc1/miR-17-92 cluster expression was not altered in CF neutrophils or plasma relative to non-CF samples, but its expression in CF sputum showed a significant positive correlation with pulmonary exacerbations and a significant negative correlation with lung function [68].

Lung fibrosis

Chronic inflammation in CF progressively destroys healthy lung tissue via fibrosis, or scarring, of the lungs. Further investigations on the effects of increased miR-155 in CF epithelial cells identified RPTOR (regulatory-associated protein of mTOR, complex 1) to be a direct target mRNA. RPTOR inhibition caused activation of TGF-β signalling and induced fibrosis via increased CTGF (connective tissue growth factor) levels [69].

Ion conductance

There have been numerous studies detailing the miRNA-mediated regulation of CFTR expression, the first of which identified the ability of miR-145 and miR-494 to directly target the 3′ UTR of the gene transcript [70]. Several subsequent studies by other groups confirmed miR-494 as a CFTR regulator [7173], and miR-101 and -144 were additionally identified as cognate miRNAs [71,74]. Oglesby et al. validated CFTR as a direct target of miR-145, -223 and -494, and showed their expression to be increased in CF bronchial brushings and furthermore in p.Phe508del CFTR homozygotes versus heterozygotes [72]. A report by Amato et al. [75] described a mutation in the CFTR 3′UTR which increased the binding affinity for miR-509-3p, leading to a reduction in CFTR protein. Further work by the same group demonstrated that use of a small peptide nucleic acid to inhibit miR-509-3p was successful in restoring CFTR levels [76]. Both miR-509-5p and miR-494 were further studied by Ramachandran et al. [73], where they were found to be up-regulated by pro-inflammatory stimuli such as tumour necrosis factor alpha, IL-1β and S. aureus via NF-κB signalling.

Although not a direct inhibitor of CFTR expression, miR-138 has been shown to target the transcription regulator SIN3A, and modulate CFTR levels in this way [77]. Overexpression of miR-138 mediated an enhancement in the number of CFTR molecules reaching the cell surface, suggesting this could be a potential avenue to explore for CF therapy [77]. Several recent studies have also demonstrated the exploitation of oligonucleotide sequences to specifically bind and inhibit miR-101, -145 and 509-3p, successfully resulting in rescue of CFTR protein levels [7881]. A miR-16 mimic was also effective in restoring p.Phe508del CFTR function, with evidence of a role for suppressed levels of the chaperone protein HSP90 (heat shock protein 90) in the mechanism [82]. Taken together, these studies offer the exciting prospect that miRNA-based therapies which enhance CFTR expression may have the capacity to increase the efficacy of the current CFTR modulators being trialled in CF patients, as miRNAs act upstream of correctors and potentiators.

In addition to the CFTR ion channel, the calcium-activated chloride ion channel ANO1 (anoctamin 1) is also down-regulated in individuals with CF and contributes to disease severity [83]. A recent publication by Sonneville et al. [84] revealed that miR-9 targets the 3′UTR of ANO1 to reduce its expression and regulate chloride activity. miR-9 was found to be increased in the CFBE41o cell line and correlated with lower levels of ANO1; this significant correlation was also observed in fully differentiated bronchial cells from CF patients [84]. Moreover, the authors showed that preventing miR-9 from binding to the 3′UTR of ANO1 restored the mucus dynamics in CF mice [84], suggesting once again that miRNA-based therapies can be envisaged for the future treatment of CF.

Unfolded protein response

Given that mutations of CFTR result in misfolding of the protein and its accumulation in the ER, ER stress and the triggering of the unfolded protein response (UPR) signalling network are considered important in CF. ATF6 (activating transcription factor 6) is a transcription factor that resides in the ER and functions as part of the UPR. ATF6 levels were found decreased in CF bronchial brushings, and miR-221, which was validated to target ATF6, was measured at elevated levels [85].

Table 1 provides a synopsis of the main miRNAs and cognate targets that are known to be dysregulated in CF, while Figure 2 illustrates their proven and/or putative roles in the pathophysiology of the disease.

Role of miRNAs and their target mRNAs in the pathophysiology of CF.

Figure 2.
Role of miRNAs and their target mRNAs in the pathophysiology of CF.

Bronchial epithelial cells are depicted in pink and macrophages in light blue. MiRNAs that were found up-regulated or down-regulated in CF versus non-CF are reported in red or green, respectively.

Figure 2.
Role of miRNAs and their target mRNAs in the pathophysiology of CF.

Bronchial epithelial cells are depicted in pink and macrophages in light blue. MiRNAs that were found up-regulated or down-regulated in CF versus non-CF are reported in red or green, respectively.

Table 1
miRNAs dysregulated in CF
miRNA ↑ or ↓ Human cell type/animal model Target Functional relevance Reference 
miR-126 ↓ Primary bronchial brushings; 16HBE14o, CFBE41o TOM1 TLR2, TLR4, IL-1R1 pathways Oglesby et al. [57
miR-155 ↑ IB3-1, S9; primary bronchial epithelial cells; primary neutrophils SHIP1

RPTOR 
PI3K/Akt pathway
Lung fibrosis 
Bhattacharyya et al. [58]

Tsuchiya et al. [69
miR-93  P. aeruginosa-infected IB3-1, CuFi-1 and NuLi-1 IL-8 Inflammation Fabbri et al. [60
miR-17 

↑ 
Primary bronchial brushings; βENaC-transgenic mice lung lysates
Primary macrophages; bone marrow-derived macrophages from F508del mice 
IL-8

ATG7, ATG16L1 
Inflammation

Autophagy 
Oglesby et al. [61]

Tazi et al. [67
miR-199a-5p ↑ Bone marrow-derived macrophages from Cftr-deficient mice CAV1 TLR4 and PI3K/Akt signalling Zhang et al. [62
miR-145 ↑ Primary nasal epithelial cells
Primary bronchial brushings; 16HBE14o, CFBE41o; primary airway epithelial cells 
SMAD3
CFTR 
TGF-β pathway
Ion conductance 
Megiorni et al. [63]
Gillen et al. [70], Oglesby et al. [72], Lutful Kabir et al. [80
miR-224-5p ↑ Primary monocytes SMAD4 TGF-β pathway McKiernan et al. [64
miR-31 ↓ Primary bronchial brushings; 16HBE14o, CFBE41o IRF-1 Protease activity Weldon et al. [65
miR-181b ↑ Primary macrophages; 16HBE14o, CFBE41o ALX/FPR2 Phagocytosis Pierdomenico et al. [66
miR-494
miR-223

miR-509-3p 



↑ 
Primary bronchial brushings; 16HBE14o, CFBE41o

Well-differentiated primary epithelia; freshly dissociated airway epithelial cells 
CFTR


CFTR 
Ion conductance


Ion conductance 
Gillen et al. [70], Megiorni et al. [71]
Oglesby et al. [72]
Ramachandran et al. [73], Amato et al. [75
miR-9 ↑ 16HBE14o, CFBE41o ANO1 Ion conductance Sonneville et al. [84
miR-221 ↑ Primary bronchial brushings; 16HBE14o, CFBE41o; βENaC-Tg mice airway tissue ATF6 UPR Oglesby et al. [85
miRNA ↑ or ↓ Human cell type/animal model Target Functional relevance Reference 
miR-126 ↓ Primary bronchial brushings; 16HBE14o, CFBE41o TOM1 TLR2, TLR4, IL-1R1 pathways Oglesby et al. [57
miR-155 ↑ IB3-1, S9; primary bronchial epithelial cells; primary neutrophils SHIP1

RPTOR 
PI3K/Akt pathway
Lung fibrosis 
Bhattacharyya et al. [58]

Tsuchiya et al. [69
miR-93  P. aeruginosa-infected IB3-1, CuFi-1 and NuLi-1 IL-8 Inflammation Fabbri et al. [60
miR-17 

↑ 
Primary bronchial brushings; βENaC-transgenic mice lung lysates
Primary macrophages; bone marrow-derived macrophages from F508del mice 
IL-8

ATG7, ATG16L1 
Inflammation

Autophagy 
Oglesby et al. [61]

Tazi et al. [67
miR-199a-5p ↑ Bone marrow-derived macrophages from Cftr-deficient mice CAV1 TLR4 and PI3K/Akt signalling Zhang et al. [62
miR-145 ↑ Primary nasal epithelial cells
Primary bronchial brushings; 16HBE14o, CFBE41o; primary airway epithelial cells 
SMAD3
CFTR 
TGF-β pathway
Ion conductance 
Megiorni et al. [63]
Gillen et al. [70], Oglesby et al. [72], Lutful Kabir et al. [80
miR-224-5p ↑ Primary monocytes SMAD4 TGF-β pathway McKiernan et al. [64
miR-31 ↓ Primary bronchial brushings; 16HBE14o, CFBE41o IRF-1 Protease activity Weldon et al. [65
miR-181b ↑ Primary macrophages; 16HBE14o, CFBE41o ALX/FPR2 Phagocytosis Pierdomenico et al. [66
miR-494
miR-223

miR-509-3p 



↑ 
Primary bronchial brushings; 16HBE14o, CFBE41o

Well-differentiated primary epithelia; freshly dissociated airway epithelial cells 
CFTR


CFTR 
Ion conductance


Ion conductance 
Gillen et al. [70], Megiorni et al. [71]
Oglesby et al. [72]
Ramachandran et al. [73], Amato et al. [75
miR-9 ↑ 16HBE14o, CFBE41o ANO1 Ion conductance Sonneville et al. [84
miR-221 ↑ Primary bronchial brushings; 16HBE14o, CFBE41o; βENaC-Tg mice airway tissue ATF6 UPR Oglesby et al. [85

lncRNA studies in cystic fibrosis

There are much fewer studies involving the expression and function of lncRNAs in CF. In the first study of its kind in CF, a microarray profile of 30 586 lncRNAs identified 1063 lncRNAs which were differentially expressed between bronchial brushings from CF versus non-CF patients [86]. XIST, centrally involved in the process of X-chromosome inactivation in females, was validated by qPCR as being up-regulated in CF samples [86]. Furthermore, TLR8-AS1 (the natural antisense lncRNA to TLR8 mRNA) was also chosen for validation, and despite very low/undetectable expression levels of the transcript it appeared to be reduced in CF cells. This was in accordance with increased expression of TLR8 mRNA [86].

Most recently, Balloy et al. compared the transcriptomic datasets of primary CF and non-CF bronchial epithelial cells at 0, 2, 4 and 6 h post infection with P. aeruginosa [87]. The analysis revealed differential expression of 25, 73, 15 and 26 lncRNA transcripts between CF versus non-CF cells at the respective time points. Two transcripts were selected for validation by qPCR; the results confirmed that MEG9 (maternally expressed 9) and BLACAT1 (bladder cancer-associated transcript 1) were down-regulated in CF cells infected with P. aeruginosa at 2 and 6 h post-infection. The authors discussed the potential implications of these findings for P. aeruginosa infection of the CF airway [87].

In another study, the lncRNA BGas was reported to function in transcriptional repression of CFTR expression [88]. BGas is expressed on the antisense strand of the CFTR gene and mediates its effects by working with other proteins to alter the local chromatin and DNA architecture of intron 11 [88]. Suppression of BGas was able to increase CFTR expression levels and ion function, and may therefore represent an exciting new target for therapeutic development.

Concluding remarks

The past 10 years has seen a significant number of investigations into the expression and function of miRNAs in CF, particularly with respect to the lung disease. Less work has been carried out in the area of lncRNA and CF; however, more reports on this topic are guaranteed in the short to medium term. Examination of the expression of other classes of ncRNA, such as circRNA, and their potential contribution to the pathophysiology of CF are not yet available; nonetheless, they are likely to be forthcoming in the not too distant future. Currently, neither ncRNA expression profiles nor expression levels of individual ncRNAs are employed in clinical practice for diagnosis or management of CF. However, there have been many recent reports suggesting that miRNAs in particular could represent potential biomarkers for CF-related disease. For example, a panel of circulating miRNAs can differentiate stages of fibrosis in CF-related liver disease patients and may be useful to complement CF liver disease-screening strategies [89]. Montanini et al. have implicated altered miRNA expression in glucose tolerance and onset of CF-related diabetes, a frequent co-morbidity in CF [90]. Given the importance of exacerbations in the decline in lung function in CF, the identification of sputum biomarkers that can reflect respiratory status is highly desirable; sputum Mirc1/Mir17-92 cluster expression has been suggested to be a useful biomarker in this regard [68]. In time, robust ncRNA biomarker panels may be identified and used clinically to enhance the management of CF.

Moreover, the encouraging in vitro and in vivo studies presented here provide a strong basis for the development of novel therapeutic approaches based on our growing knowledge of the functions of specific ncRNAs, especially miRNAs, in the pathogenesis of CF lung disease. Within the next decade it is possible that CF-specific therapies based on either enhancing or inhibiting ncRNA expression or function could become a reality.

Abbreviations

     
  • ALX/FPR2

    lipoxin A4 receptor/formyl-peptide receptor type 2

  •  
  • ANO1

    anoctamin 1

  •  
  • ATF6

    activating transcription factor 6

  •  
  • BLACAT1

    bladder cancer-associated transcript 1

  •  
  • CF

    cystic fibrosis

  •  
  • CFTR

    cystic fibrosis transmembrane receptor

  •  
  • circRNA

    circular RNA

  •  
  • CTGF

    connective tissue growth factor

  •  
  • CTSS

    cathepsin S

  •  
  • ENaC

    epithelium sodium channel

  •  
  • ER

    endoplasmic reticulum

  •  
  • HSP90

    heat shock protein 90

  •  
  • IRF-1

    interferon regulatory factor 1

  •  
  • lincRNA

    long intervening/intergenic non-coding RNA

  •  
  • lncRNA

    long non-coding RNA

  •  
  • MEG9

    maternally expressed 9

  •  
  • miRNA

    micro RNA

  •  
  • NAT

    natural antisense transcript

  •  
  • ncRNA

    non-coding RNA

  •  
  • NF-κB

    nuclear factor kappa-light-chain-enhancer of activated B cells

  •  
  • PI3K

    phosphatidylinositol 3-kinase

  •  
  • piRNA

    piwi-interacting RNA

  •  
  • RPTOR

    regulatory-associated protein of mTOR, complex 1

  •  
  • SHIP1

    Src homology-2 domain-containing inositol 5-phosphatase 1

  •  
  • snoRNA

    small nucleolar RNA

  •  
  • snRNA

    small nuclear RNA

  •  
  • TGF-β

    transforming growth factor beta

  •  
  • TLR

    Toll-like receptor

  •  
  • TNF-α

    tumour necrosis factor alpha

  •  
  • TOM1

    target of Myb1

  •  
  • TTP

    tristetraprolin

  •  
  • UPR

    unfolded protein response

  •  
  • UTR

    untranslated region

  •  
  • XIST

    X inactive-specific transcript

Author Contribution

All the authors wrote the article and approved the final version.

Funding

Research funding in the Greene group is gratefully acknowledged from Cystic Fibrosis Foundation Therapeutics (GREENE-15XXO), Horizon2020 (MSCA-IF award 707771 GENDER-CF to A Glasgow), and the Irish Research Council [GOIPG/2015/2393].

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors contributed equally to this work.