**2. Regulation of CFTR expression**

phenylalanine at position 508 of the protein, and the mutated protein is retained in the endoplasmic reticulum and rapidly degraded via the endoplasmic reticulum-associated degradation pathway. Up to now, almost 2000 CFTR mutations have been identified (http:// genet.sickkids.on.ca). A good correlation can generally be observed between *CFTR* genotype and the gastrointestinal disease, the pancreatic status, and the reproductive tract abnormali‐ ties, but the lung disease outcome is difficult to predict based solely on *CFTR* genotype (http:// www.cftr2.org/). For example, it has been shown that siblings and monozygous twins, thus carrying the same *CFTR* genotype, even if living in the same environment and receiving the same medical care, may develop different spatial and temporal patterns in lung disease progression. The basis for variability in severity of CF lung disease is poorly understood and depends on concomitant expression of other genetic and environmental factors. Over the recent years, exploring the role of genetics/genomics (e.g. modifier genes, gene-environment interactions, epigenetics, etc.) has received growing attention in CF, with the aim of unveiling basic mechanisms and bringing in better understanding of the pathophysiology of the disease, helping to predict its progression, and hopefully leading to specially designed novel thera‐

Of the whole human genome, only a small fraction, the protein-coding part, has long attracted attention because of the pervasive role of genes in determining amino acid sequences of expressed proteins, leading to observable consequences of mutations. Later on, following the discovery of transduction factors regulating gene translation, the initial view of "junk DNA", corresponding to the majority of DNA, had to be revisited. Following the identification of nonmessenger RNA functions, the new concept of a network of non-coding transcriptome that

Non-coding RNAs are presently broadly categorized into three classes. The major class (well over 90% of total RNA) makes up the so-called housekeeping RNAs; they consist of small nuclear, small nucleolar, transfer, and ribosomal RNAs, the latter interacting with protein and transfer RNA to form the functional ribosome complex. The other classes of non-coding RNAs are long (>200) and short (<200) ribonucleotides. The best characterized and most extensively studied family of non-coding RNAs is that of microRNAs (miRNAs), short (17--27 nucleotides in length) single-stranded RNA molecules, which negatively regulate the translation of messenger RNAs into proteins. Figure 1 summarizes the general mechanism of biogenesis of miRNAs. Highly phylogenetically conserved, they bind to the 3'UTR (untranslated region) of target mRNAs, thereby potently repressing the target mRNA translation into protein or favoring mRNA degradation. To date, more than 1800 mature miRNAs have been identified in the human genome (http://www.mirbase.org). Bioinformatics studies predict that miRNAs

As essential components of the regulatory system of gene expression, miRNAs have been shown to influence the development, the severity, the prognosis and/or the progression of a number of inherited diseases [1]. Differential expression studies of miRNAs have evidenced an impact on lung disease development in chronic obstructive pulmonary disease (COPD), asthma, lung inflammation, consequences of smoke exposure and airway allergy in human and in animal models of the diseases [2]. Since marked inflammation is a major feature of CF

peutic strategies.

234 Cystic Fibrosis in the Light of New Research

regulates protein-coding expression emerged.

potentially regulate the expression of about 60% of human genes.

The human *CFTR* gene is located on chromosome 7 and spans 189 kb. Its mRNA transcript is 6.2kb long and includes a 1.5kb 3'UTR containing multiple potential binding sites for miRNAs. According to computational analyses using common bioinformatic programs such as Tar‐ getScan (http://www.targetscan.org), PicTar (http://pictar.mdc-berlin.de), and miRanda (http://www.microrna.org), CFTR 3'UTR contains almost 500 putative miRNA target sites, several of which have already been examined.

The main miRNAs involved in cystic fibrosis pathogenesis are summarized in figure 2.

Gillen et al. were the first to experimentally show that miRNAs could regulate CFTR expression [3]. Among the numerous miRNAs putatively repressing CFTR mRNA, twelve miRNAs (miR-145, miR-331-3p, miR-376a/b, miR-377, miR-384, miR-494, miR-600, miR-607, miR-939, miR-1246, miR-1290 and miR-1827) were able to decrease CFTR mRNA levels in the human Bronchial epithelial (HBE) 16HBE-14o-, colon carcinoma epithelial Caco-2 and the pancreatic adenocarcinoma PANC-1 cell lines. Direct targeting of the CFTR 3'UTR was evidenced for miR-145 and miR-494 in Caco-2 cells. The results [3] showed that the pattern of miRNAs effects on targeted CFTR 3'UTR varies with cell lines, suggesting that miRNA-driven gene expression could be tissue specific. Another study [4] demonstrated direct repression of CFTR expression by miR-101 and miR-494 in the HEK (human embryonic kidney) cell line expressing CFTR 3'UTR constructs. miR-101 and miR-494 seem to bind directly to the CFTR 3'UTR at positions 1508–1514 and 1140–1147, respectively. Downregulation of CFTR gene expression by miR-101 as well as by miR-144 and miR-145 was further confirmed in the 16HBE14o- cell line and in primary human airway cells, which are relevant models of CF lung disease [5, 6].

In a non-CF context, expression of miR-101 and miR-144 was previously shown to be upre‐ gulated in lungs upon exposure to air pollutants (such as cigarette smoke or cadmium) and correlated with loss of CFTR expression [4]. Hassan et al. [5] demonstrated upregulation of miR-101 in vivo in mice exposed to cigarette smoke, a condition associated with acquired loss of CFTR function [7]. miR-101 was also found highly expressed in lungs of COPD smoking patients in comparison with that of healthy non-smoking subjects. Moreover, miRNA expres‐ sion profile analysis performed in the CF bronchial brushings in comparison to healthy controls showed high levels of miR-101, thus further reinforcing the potential role of miR-101 in CF [8]. Interestingly, synergistic effects between miR-101 and miR-494 were observed, although they are targeting different sites in CFTR 3'UTR [4]. Similar synergy was found for miR-509–3p and miR-494 [9]. Therefore, distinct miRNAs may act cooperatively to regulate CFTR expression and function in primary airway epithelial cells.

These miRNAs are likely able to modulate F508del-CFTR mRNA expression as well. Interest‐ ingly, increased expression of miR-145, miR-223 and miR-494 in vivo has been shown to correlate with decreased CFTR expression in bronchial epithelium of individuals bearing the F508del-CFTR mutation [3] as well as in CFBE41o- cells [10]. Experimental modulation of miRNA expression confirmed the hypothesis supporting the view that deregulation of miRNA may affect CFTR biogenesis in CF cells. Another study demonstrated that miRNA can indirectly influence CFTR biogenesis by modulating expression of other regulatory elements such as transcription factors [11]. It has been shown that modulating the transcription regulation factor SIN3A expression with miR-138 mimics increased biogenesis and cell surface expression of both wild-type and F508del-CFTR proteins. Interestingly, miR-138 indirectly prevented proteosomal degradation of F508del-CFTR mutant and favored its trafficking towards the apical membrane in HBEs from CF patients [11].

According to computational analyses using common bioinformatic programs such as Tar‐ getScan (http://www.targetscan.org), PicTar (http://pictar.mdc-berlin.de), and miRanda (http://www.microrna.org), CFTR 3'UTR contains almost 500 putative miRNA target sites,

Gillen et al. were the first to experimentally show that miRNAs could regulate CFTR expression [3]. Among the numerous miRNAs putatively repressing CFTR mRNA, twelve miRNAs (miR-145, miR-331-3p, miR-376a/b, miR-377, miR-384, miR-494, miR-600, miR-607, miR-939, miR-1246, miR-1290 and miR-1827) were able to decrease CFTR mRNA levels in the human Bronchial epithelial (HBE) 16HBE-14o-, colon carcinoma epithelial Caco-2 and the pancreatic adenocarcinoma PANC-1 cell lines. Direct targeting of the CFTR 3'UTR was evidenced for miR-145 and miR-494 in Caco-2 cells. The results [3] showed that the pattern of miRNAs effects on targeted CFTR 3'UTR varies with cell lines, suggesting that miRNA-driven gene expression could be tissue specific. Another study [4] demonstrated direct repression of CFTR expression by miR-101 and miR-494 in the HEK (human embryonic kidney) cell line expressing CFTR 3'UTR constructs. miR-101 and miR-494 seem to bind directly to the CFTR 3'UTR at positions 1508–1514 and 1140–1147, respectively. Downregulation of CFTR gene expression by miR-101 as well as by miR-144 and miR-145 was further confirmed in the 16HBE14o- cell line and in

The main miRNAs involved in cystic fibrosis pathogenesis are summarized in figure 2.

primary human airway cells, which are relevant models of CF lung disease [5, 6].

CFTR expression and function in primary airway epithelial cells.

In a non-CF context, expression of miR-101 and miR-144 was previously shown to be upre‐ gulated in lungs upon exposure to air pollutants (such as cigarette smoke or cadmium) and correlated with loss of CFTR expression [4]. Hassan et al. [5] demonstrated upregulation of miR-101 in vivo in mice exposed to cigarette smoke, a condition associated with acquired loss of CFTR function [7]. miR-101 was also found highly expressed in lungs of COPD smoking patients in comparison with that of healthy non-smoking subjects. Moreover, miRNA expres‐ sion profile analysis performed in the CF bronchial brushings in comparison to healthy controls showed high levels of miR-101, thus further reinforcing the potential role of miR-101 in CF [8]. Interestingly, synergistic effects between miR-101 and miR-494 were observed, although they are targeting different sites in CFTR 3'UTR [4]. Similar synergy was found for miR-509–3p and miR-494 [9]. Therefore, distinct miRNAs may act cooperatively to regulate

These miRNAs are likely able to modulate F508del-CFTR mRNA expression as well. Interest‐ ingly, increased expression of miR-145, miR-223 and miR-494 in vivo has been shown to correlate with decreased CFTR expression in bronchial epithelium of individuals bearing the F508del-CFTR mutation [3] as well as in CFBE41o- cells [10]. Experimental modulation of miRNA expression confirmed the hypothesis supporting the view that deregulation of miRNA may affect CFTR biogenesis in CF cells. Another study demonstrated that miRNA can indirectly influence CFTR biogenesis by modulating expression of other regulatory elements such as transcription factors [11]. It has been shown that modulating the transcription regulation factor SIN3A expression with miR-138 mimics increased biogenesis and cell surface expression of both wild-type and F508del-CFTR proteins. Interestingly, miR-138 indirectly

several of which have already been examined.

236 Cystic Fibrosis in the Light of New Research

Altogether, these data are in favor of a role for several miRNAs in the post-transcriptional regulation of the CFTR channel synthesis and trafficking. Because several of them are deregu‐ lated in CF, they could play a major role in CF lung pathology.
