Regulation of Cystic Fibrosis Transmembrane Conductance Regulator by MicroRNA-145, -223, and -494 Is Altered in ΔF508 Cystic Fibrosis Airway Epithelium

Abstract

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is altered in individuals with the ΔF508 CFTR mutation. We previously reported differential expression of microRNA (miRNA) in CF airway epithelium; however, the role of miRNA in regulation of CFTR expression here remains unexplored. In this study, we investigated the role of upregulated miRNAs in CFTR regulation in vivo in bronchial brushings from individuals homozygous or heterozygous for ΔF508 CFTR, validated our observations in vitro, and assessed the impact of defective chloride ion conductance, genotype, and colonization status on miRNA expression. miRNA target prediction was performed in silico, and expression of miRNA and target genes were measured by quantitative real-time PCR and/or Western blotting. Overexpression and inhibition studies were performed with pre-miRs or antimiRs, respectively, and a luciferase reporter gene was used to elucidate direct miRNA–target interactions. miR-145, miR-223, and miR-494 were upregulated in CF versus non-CF bronchial brushings and cell lines; in ΔF508 CFTR homozygotes versus heterozygotes; in subjects positive for P. aeruginosa; and in cells treated with a CFTR inhibitor or IL-1β. Reciprocal downregulation or upregulation of CFTR gene and/or protein expression was observed after miRNA manipulation and direct miRNA–target relationships demonstrated via a reporter system containing a wild type or mutated full-length CFTR 3′ untranslated region. Increased expression of miR-145, miR-223, and miR-494 in vivo in bronchial epithelium of individuals carrying the ΔF508 CFTR mutation correlates with decreased CFTR expression. Defective CFTR function, Pseudomonas colonization, and inflammation may affect miRNA expression and contribute to the regulation of ΔF508 CFTR.

Developmental control of CFTR: from bioinformatics to novel therapeutic approaches

“…The data confirms previous studies implicating miR-145 and miR-101 as important modulators of CFTR expression [5,6,8,9] and build on them by demonstrating how MBBOs based on these miRNAs can affect CFTR gene expression and CFTR protein function. The authors suggest that the MBBOs may be developed as tools for CFTR correction in people with CF.…”

“…This will be particularly important for the further development of MBBOs as therapeutics for CF. Previous studies have reported how infection and inflammation affect the expression of miRNAs, including those that regulate CFTR [9,13]. This suggests that higher than normal levels of MBBOs may be required to ensure an inhibitory effect on endogenous miRNA activity.…”

Long noncoding RNA are aberrantly expressed in vivo in the cystic fibrosis bronchial epithelium

“…Performed on endobronchial brushings from people with and without CF, microRNA profiling studies identified various microRNAs that had altered expression in CF. Our group also identified that three of these microRNAs with increased expression in vivo (miR-145, miR-223, and miR-494) correlate with decreased Phe508del CFTR expression and regulate its expression (Oglesby et al, 2013) . What remains to be seen is whether there exists an altered lncRNA profile in the CF airway epithelium.…”

miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production

“…Transfections were performed using GeneJuice (Novagen, Madison ,WI, USA) for plasmid DNA and RiboJuice (Novagen) for miRNA in OptiMEM reduced serum media (Life Technologies), as recommended. AntimiRs do not always degrade their target miRNA and we measured variable antimiR-specific target miRNA knockdown of 20-80% using transfection conditions that were optimised previously to ensure uniform transfection across all wells [27]. Lysates were prepared 36 h post-transfection and luciferase activities were measured using the Luciferase Assay System (Promega) and coelenterazine (Marker Gene Technologies, Eugene, OR, USA).…”

“…IB3 and S9 cells were grown in LHC-8 medium (Gibco) with 5% FCS and 1% Pen-Strep. In some experiments S9 cells were grown to a density of 5×10 5 cells·cm −2 and chronically stimulated (5 days) [31] with 1% Pseudomonas-conditioned medium (PCM) [27] in LHC-8 medium (Gibco) with 1% FCS and 1% Pen-Strep. Media were changed every day.…”