Cystic fibrosis results from reduced cystic fibrosis transmembrane conductance regulator protein activity leading to defective epithelial ion transport. Ca2+-activated Cl− channels mediate physiological functions independently of cystic fibrosis transmembrane conductance regulator. Anoctamin 1 (ANO1/TMEM16A) was identified as the major Ca2+-activated Cl− channel in airway epithelial cells, and we recently demonstrated that downregulation of the anoctamin 1 channel in cystic fibrosis patients contributes to disease severity via an unknown mechanism. Here we show that microRNA-9 (miR-9) contributes to cystic fibrosis and downregulates anoctamin 1 by directly targeting its 3′UTR. We present a potential therapy based on blockage of miR-9 binding to the 3′UTR by using a microRNA target site blocker to increase anoctamin 1 activity and thus compensate for the cystic fibrosis transmembrane conductance regulator deficiency. The target site blocker is tested in in vitro and in mouse models of cystic fibrosis, and could be considered as an alternative strategy to treat cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal genetic disease, caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. CF is characterized by an ionic imbalance and thickened mucus, which impair mucociliary clearance and promote bacterial colonization and the establishment of infection/inflammation cycles. However, the origin of this inflammation remains unclear, although microRNAs (miRNAs) are suspected to be involved. MiRNAs are small non-coding RNAs that bind to the 3'-untranslated regions (UTRs) of target gene mRNA, thereby repressing their translation and/or inducing their degradation. The goal of this study was to investigate the role of microRNAs associated with pulmonary inflammation in CF patients. Through the analysis of all miRNAs (miRNome) in human primary air-liquid interface cultures, we demonstrated that miR-199a-3p is the only miRNA downregulated in CF patients compared to controls. Moreover, through RNA sequencing (transcriptome) analysis, we showed that 50% of all deregulated mRNAs are linked directly or indirectly to the NF-κB pathway. To identify a specific target, we used bioinformatics analysis to predict whether miR-199a-3p targets the 3'-UTR of IKBKB, which encodes IKKβ, a major protein in the NF-κB pathway. Subsequently, we used bronchial explants from CF patients to show that miR-199a-3p expression is downregulated compared to controls and inversely correlated with increases in expression of IKKβ and IL-8. Through functional studies, we showed that miR-199a-3p modulates the expression of IKBKB through a direct interaction at its 3'-UTR in bronchial epithelial cells from CF patients. In miR-199a-3p overexpression experiments, we demonstrated that for CF cells, miR-199a-3p reduced IKKβ protein expression, NF-κB activity, and IL-8 secretion. Taken together, our findings show that miR-199a-3p plays a negative regulatory role in the NF-κB signalling pathway and that its low expression in CF patients contributes to chronic pulmonary inflammation. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.