**5. miRNAs as biomarkers and potential therapies**

miRNAs might non-specifically regulate epithelial differentiation in CF cells and could be

**Figure 2.** Main miRNAs involved in cystic fibrosis pathogenesis The left part depicts miRNA-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis and trafficking. The right part zooms into the regulation of interleukin (IL)-8 production by miRNAs. miRNAs which may drive phenotypic changes on multicilio‐ genesis, airway remodeling and disorganization of tight junction are also represented. Red arrows depict changes re‐ ported in CF; up arrows indicate upregulated miRNAs and phenotype; and down arrows indicate miRNA and

MicroRNAs potentially altered and phenotypic consequences in CF are summarized in

**Target MicroRNA Expression Consequences References**

Downregulation of CFTR

Downregulation of CFTR

Inhibition of proteosomal degradation of F508del-CFTR, Cl-

Unexplained downregulation of

4,5,7,8,10

3,5,9,10

11

8

expression

expression

permeability

NF-κB

Overexpressed in CF nasal and

Overexpressed in CF bronchial brushings and CFBE41o- cell line

SIN3A miR-138 Corrector of CFTR trafficking

bronchial brushing and CFBE41o-

bronchial biopsies

phenotype downregulation. CSE: cigarette smoke exposure; CTS: cathepsin.

Table 1.

CFTR miR-101, -144, -494

CFTR miR-145,-223,


TOM1 miR-126 Decreased expression in CF

cell line

involved in loss of epithelial polarity.

240 Cystic Fibrosis in the Light of New Research

As in other diseases in which miRNAs have been involved, such as asthma, COPD, idiopathic pulmonary fibrosis, cancer and diabetes, further analyses of miRNA expression-function relationships will very likely reveal new genetic factors that could be targeted in therapy. There is also a great hope that miRNAs could be used as diagnostic markers and represent new prognostic factors that might influence the course of the CF disease.

The pathophysiology of CF is very variable from patient to patient and is only partly explained by the *CFTR* genotype. Two recent studies raised the hypothesis that profiling serum miR‐ Nome could identify miRNAs as potential prognostic biomarkers [16, 36]. First, elevated miR-155 serum levels have been detected in patients with CF, possibly reflecting its high expression in CF airway cells [16]. Second, a prototype study by Cook et al. has suggested that serum miRNAs could be used as diagnostic markers in CF liver disease [37]. Profiles of circulating miRNA levels in patients with CF liver disease were compared to those of CF patients without liver disease and of non-CF controls. For the first time, changes in circulating miRNA levels were identified in CF and they were correlated with disease status, suggesting that serum miRNA analysis may help predict early onset of hepatic fibrosis in CF [36]. It could be expected that new biomarkers of the course of the CF lung disease will be identified in the coming years.

It can also be foreseen that single nucleotide polymorphisms (SNPs) in the 3'UTR of miRNAtargeted genes, in particular CFTR, may explain phenotype variability. As an illustration, an SNP (c.\*1043A>C) was identified in the 3'UTR of CFTR in a patient with a CFTR-related disease [38]. CFTR-related diseases are clinical entities associated with CFTR dysfunction but that do not fulfil diagnostic criteria for classical CF (e.g., Congenital Bilateral Absence of Vas Deferens (CBAVD), chronic pancreatitis and disseminated bronchiectasis). This SNP was located within the binding site of two miRNAs including miR-509-3p (shown otherwise to directly target CFTR mRNA), and experimental data suggested that it might impair the regulation of gene expression. That could explain the mild phenotype, as this SNP would act as a mild mutation. Consequently, polymorphisms in the CFTR 3'UTR may play a role in the observed heteroge‐ neous phenotype. Molecular analysis of the 3'UTR of CFTR could therefore be performed as a differential diagnostic tool in patients presenting with suggestive clinical symptoms of CF but no mutation in the *CFTR* gene per se.

Modulating miRNA expression in vivo looks very appealing for developing new CF therapies [38]. Indeed miRNAs are short and need to be delivered only to the cytoplasm, as opposed to nucleus delivery required for DNA-based constructs. Moreover, miRNA-based therapies have several advantages over gene therapy strategies aiming at restoring CFTR expression. First, miRNA modulators are likely to target multiple genes in the context of a deregulated network. However, this might also be a major drawback as potential off-target effects may cause adverse phenotypes. Second, as in all cases of gene therapies, tissue-specific delivery remains a major issue in miRNA-based therapies. An interesting approach has been used by McKiernan et al. to successfully deliver miRNA replacement therapy in CFBE41o- cells [39]. They demonstrated that polyethyleneimine nanoparticles complexed with pre-miR-126 resulted in significant knock-down of TOM1, previously described as a direct target of miR-126 [8]. This result shows that polymeric nanoparticles may be used to effectively deliver miRNA replacement therapy with no adverse effects and may present a strong advantage in comparison to virus-based delivery strategies. As for any other disease for which miRNA-based therapy is considered as an attractive new option, factors controlling the stability of the miRNAs, the delivery systems and the off-target effects of miRNA-based therapies represent strong challenges for the future of development of such drugs.
