RPTOR, a novel target of miR-155, regulates the fibrotic phenotype of cystic fibrosis lung epithelium by upregulating CTGF

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the most frequent of which is F508del-CFTR. CF is characterized by excessive secretion of pro-inflammatory chemokines into the airway lumen, inducing a highly inflammatory cellular phenotype. This process triggers fibrosis, causing airway destruction and leading to high morbidity and mortality. We previously reported that miR-155 is up-regulated in CF lung epithelial cells, but the molecular mechanisms by which miR-155 affects the disease phenotype is not understood. Here we report that the protein RPTOR (regulatory associated protein of mTOR, complex 1) is a novel target of miR-155 in CF lung epithelial cells. The suppression of RPTOR expression and subsequent activation of TGF-β signaling resulted in the induction of fibrosis by up-regulation of connective tissue growth factor (CTGF) in CF lung epithelial cells. Thus, we propose that miR-155 can regulate fibrosis of CF lungs through the RPTOR-TGF-β-CTGF axis, highlighting its potential value in CF therapy. Elevated expression of miR-155 in CF bronchial epithelial cells induces expression of the pro-inflammatory cytokine IL8 via increased PI3K/Akt signaling 10, and we sought to identify miR-155 targets that regulate the CF disease phenotype. Two experimental strategies were employed in a concurrent gene expression study: in the first, a 3'-end-biotinylated miR-155 (N=3) or biotinylated cel-miR-67 control (N=2) was transfected into IB3-1/S9 lung epithelial cells. Streptavidin bead-based affinity purification was used to enrich cell lysates for mRNA:miR-155 complexes, which were extracted and compared using microarray analysis to the cel-miR-67 control. In the second, IB3-1/S9 cells were incubated with either a miR-155 (N=2) or a control mimic (N=2). From the microarray analysis, potential mRNA targets of miR-155 are expected to be enriched in the affinity-based isolation employing biotinylated-miR-155, and should be down-regulated in cells where miR-155 is overexpressed. Thus, we identified genes with positive Z-ratio from the former group (upregulated mRNAs that bind miR-155) and with negative Z-ratio from the latter group (transcripts that are downregulated if miR-155 is overexpressed).