*3.2.2. Functional approaches*

To evaluate *in vitro* the impact of miRNAs on *CFTR* gene regulation, several approaches can be used (Figure 5 A). a) First of all, *in silico* analysis by using bioinformatics tools can be performed (see 3.2.1). b) The role of specific miRNAs could be assessed *in vitro* by using airway cell lines and luciferase gene reporter assays, in which the luciferase coding sequence is under the control of the 3' UTR of *CFTR,* as previously described [11,3]. c) By transfecting mimics or precursors, miRNAs can be overexpressed and the relative level of luciferase expression reflects the effect of a given miRNA on the 3'UTR-*CFTR*. Inhibitors may also be used to confirm the specific effect of the miRNA under study. d) To validate the direct effect of the identified miRNAs (for instance, miRNA-494 and miRNA-101), the cis element for miRNAs binding in the 3'UTR-*CFTR* can be mutated by site-directed mutagenesis (created mutation). Target-site blockers may also be used to inhibit binding to the tested motif [3]. e) To validate these effects, the endogenous level of *CFTR* mRNA following miRNA overexpression or down-regulation can be assessed using different techniques.
