Identification of a novel functional miR-143-5p recognition element in the Cystic Fibrosis Transmembrane Conductance Regulator 3'UTR.

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs involved in regulation of gene expression. They bind in a sequence-specific manner to miRNA recognition elements (MREs) located in the 3' untranslated region (UTR) of target mRNAs and prevent mRNA translation. MiRNA expression is dysregulated in cystic fibrosis (CF), affecting several biological processes including ion conductance in the epithelial cells of the lung. We previously reported that miR-143 is up-regulated in CF bronchial brushings compared to non-CF. Here we identified two predicted binding sites for miR-143-5p (starting at residues 558 and 644) on the CFTR mRNA, and aimed to assess whether CFTR is a true molecular target of miR-143-5p. Expression of miR-143-5p was found to be up-regulated in a panel of CF vs non-CF cell lines (1.7-fold, P = 0.0165), and its levels were increased in vitro after 20 hours treatment with bronchoalveolar lavage fluid from CF patients compared to vehicle-treated cells (3.3-fold, P = 0.0319). Luciferase assays were performed to elucidate direct miRNA::target interactions and showed that miR-143-5p significantly decreased the reporter activity when carrying the wild-type full length sequence of CFTR 3'UTR (minus 15%, P = 0.005). This repression was rescued by the disruption of the first, but not the second, predicted MRE, suggesting that the residue starting at 558 was the actual active binding site. In conclusion, we here showed that miR-143-5p modestly but significantly inhibits CFTR, improving the knowledge on functional MREs within the CFTR 3'UTR. This could lead to the development of novel therapeutic strategies where miRNA-mediated CFTR repression is blocked thereby possibly increasing the efficacy of the currently available CFTR modulators.