**3. Role of non-coding RNAs in the physiological regulation of** *CFTR* **gene expression**

As the function of lncRNAs has not been studied yet, we only present findings on the miRNA roles in the regulation of *CFTR* gene expression.

### **3.1. miRNAs and** *CFTR* **gene expression**

*CFTR* gene expression is spatially and temporally regulated. Several studies have demon‐ strated the differential use of transcription start sites, depending on the tissue type or the developmental stage [52-55]. In the lung, *CFTR* transcripts can be detected early during embryo development (12th week of pregnancy) and their level progressively increases up to the 24th week of pregnancy. Thereafter, *CFTR* expression in the airways decreases and is repressed until after birth and remains very low during adult life [56,60]. The changes in CFTR protein expression in human foetuses are consistent with *CFTR* mRNA temporal pattern of expression [60]. In a recent study, we showed that miRNAs (miR-101, miR-145, miR-384) regulate the switch from strong foetal to very low *CFTR* expression after birth. Specifically, miR-101 and miR-145 negatively regulate the level of *CFTR* transcripts in adult lung cells, while they have no effect in foetal lung cells. miR-101 directly acts on its cognate site in the 3'UTR-*CFTR* in combination with an overlapping AU-rich element. Other studies showed that *CFTR* expres‐ sion is also post-transcriptionally regulated by miRNAs, such as miR-145 and miR-494 [10,11]. Gillen *et al.* demonstrated that miR-145 is expressed in primary adult human airway epithelial cells, where *CFTR* expression is low, and directly acts on *CFTR* stability [10]. In addition to its specific role in mature lung cells, miR-101 decreases luciferase activity in an embryonic kidney cell line [11], whereas it does not affect *CFTR* mRNA stability in pancreatic cell lines [10], suggesting a potential role as a tissue-specific factor.

### **3.2. Methods to investigate miRNA role in the regulation of** *CFTR* **gene expression**

Different approaches can be employed to investigate miRNA role in the regulation of *CFTR* gene expression, as depicted in Figure 5A.

**Figure 5.** Approaches for miRNA study. A. Strategies used to study the involvement of miRNAs in the regulation of *CFTR* gene expression. B. Strategies to identify miRNAs that are deregulated in CF samples compared to non-CF sam‐ ples. Chips for global miRNA profiling are commercialized by Agilent, Affimetrix and Exiqon. miR-seq (miRNA se‐ quencing) is usually performed by Illumina platforms. Luc, luciferase; TLDA, TaqMan Array Micro Fluidic Cards (Applied Biosystems); WT, wild type.

### *3.2.1. Predictive tools are freely available*

**Disease MicroRNAs Targets References**

**3. Role of non-coding RNAs in the physiological regulation of** *CFTR* **gene**

As the function of lncRNAs has not been studied yet, we only present findings on the miRNA

*CFTR* gene expression is spatially and temporally regulated. Several studies have demon‐ strated the differential use of transcription start sites, depending on the tissue type or the developmental stage [52-55]. In the lung, *CFTR* transcripts can be detected early during embryo development (12th week of pregnancy) and their level progressively increases up to the 24th week of pregnancy. Thereafter, *CFTR* expression in the airways decreases and is repressed until after birth and remains very low during adult life [56,60]. The changes in CFTR protein expression in human foetuses are consistent with *CFTR* mRNA temporal pattern of expression [60]. In a recent study, we showed that miRNAs (miR-101, miR-145, miR-384) regulate the switch from strong foetal to very low *CFTR* expression after birth. Specifically, miR-101 and miR-145 negatively regulate the level of *CFTR* transcripts in adult lung cells, while they have no effect in foetal lung cells. miR-101 directly acts on its cognate site in the 3'UTR-*CFTR* in combination with an overlapping AU-rich element. Other studies showed that *CFTR* expres‐ sion is also post-transcriptionally regulated by miRNAs, such as miR-145 and miR-494 [10,11]. Gillen *et al.* demonstrated that miR-145 is expressed in primary adult human airway epithelial cells, where *CFTR* expression is low, and directly acts on *CFTR* stability [10]. In addition to its

miR-106a IL-10 [81] miR-21 IL-12 [82] miR-133a RhoA [83] miR-26a Glycogen synthase kinase 3ß [84] let-7 IL-13 [85]

miR-181d Interferon γ, collagen XVI αI [86] miR-30c Proto-cadherin [86] miR-146a Prostaglandin E2 [87]

miR-21 SMAD7 [88] miR-155 KGF [89] miR-let7d HMGA2 [90]

Asthma

286 Cystic Fibrosis in the Light of New Research

COPD

Idiopathic pulmonary fibrosis

**expression**

COPD, chronic obstructive pulmonary disease.

**Table 1.** Examples of pulmonary diseases in which miRNAs have a role

roles in the regulation of *CFTR* gene expression.

**3.1. miRNAs and** *CFTR* **gene expression**

Predictive tools are necessary to assess the putative presence of miRNA binding motifs. A nonexhaustive list, including information about each program, is proposed in Table 2. Databases collecting all information on miRNAs are listed in Table 3. Although miRBase (http:// www.mirbase.org/) is the most used database and collects links for several predictive pro‐ grams, others exist. These tools have been developed to predict miRNA targets or the miRNAs that putatively bind to a selected gene. Some of them propose the possibility to input several miRNAs and/or several genes to identify integrated networks.



**Table 2.** Free bioinformatics tools for miRNAs

grams, others exist. These tools have been developed to predict miRNA targets or the miRNAs that putatively bind to a selected gene. Some of them propose the possibility to input several

**Name Website Characteristics References**

structure taken into account. [91]

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

Target site prediction for human, mouse, rat, fruitfly

Thermodynamic stability of RNA duplexes taken

Algorithm for target-site prediction based on the alignment of 3'UTR with predicted sites. Several databases are used for vertebrates, flies, nematodes.

hybridization for long and mainly short RNAs, such as microRNAs to one or more given targets.

Target-site prediction for human and other species that evaluates the microRNA targets accessibility as

Tool for miRNA target-site prediction and functional annotation based on the mirTarget algorithm for all known human, dog, rat, and chicken transcripts.

Target-site prediction program taking intro account both conserved and non-conserved miRNA regulatory elements (MREs) and providing scores as an indication of the expected fold change in protein

Prediction of target sites based on alignment and

Provides links to other mains programs for miRNA target prediction (e.g., TargetScan, PicTar).

Web-based system based on the analysis of conserved sequences (seed or 3' miRNAs) using external prediction tools (TargetScan, miRanda,

Analyzes miRNA biological functions using the KEGG pathway to draw a miRNA target interaction

Uses the computational tools, miRanda, RNAhybrid, and TargetScan to identify miRNA targets in the

Tool for finding the minimum free energy

miRNAs and/or several genes to identify integrated networks.

miRanda http://microrna.org/

288 Cystic Fibrosis in the Light of New Research

PicTar http://www.pictar.org/

RNAhybrid http://bibiserv.techfak.uni-

miRDB http://mirdb.org/miRDB/

miRBase Target http://www.mirbase.org/

PITA

DIANA-microT

miRTar

miRNAMap

bielefeld.de/rnahybrid/

http:// genie.weizmann.ac.il/pubs/ mir07/mir07\_data.html

http:// diana.cslab.ece.ntua.gr/ microT/

http:// mirtar.mbc.nctu.edu.tw/ human/

http:// mirnamap.mbc.nctu.edu.t w/index.php

TargetScan http://www.targetscan.org/Target site prediction for mammals. Secondary

and nematode.

into account.

a component analysis.

production.

conservation.

PITA, RNAhybrid).

3'UTR of target genes.

network.


**Table 3.** Free databases for miRNAs
