CircRNA_0026344 via exosomal miR-21 regulation of Smad7 is involved in aberrant cross-talk of epithelium-fibroblasts during cigarette smoke-induced pulmonary fibrosis

Abstract

For smoking-induced pulmonary fibrosis (PF), a serious disease endangering human health, there is no effective clinical treatment. Aberrant epithelium-fibroblast cross-talk is involved in formation of the excessive extracellular matrix (ECM) that contributes to PF. Circular RNAs have been associated with various pulmonary diseases. However, the mechanisms of circRNAs in PF are not clear. Herein, our goals were to investigate the involvement of circRNA_0026344 in the aberrant epithelium-fibroblast cross-talk induced by cigarette smoke (CS) and to define its mechanism. Chronic exposure (16 weeks) of BALB/c mice to 500 mg/m³ CS induced lung injury and fibrosis in lung tissues. From HBE cells, circRNA_0026344 was selected by microarray analysis and verified as that with the most severe down-regulation caused by cigarette smoke extract (CSE). The regulatory relationship between circRNA_0026344 and miR-21 was assessed by use of bioinformatics, RNA pull-down assays, and qRT-PCR. We found that miR-21 binding sites were present in circRNA_0026344 and, in HBE cells, it could act as a sponge for miR-21. When pcDNA3.0-circRNA_0026344, a high expression plasmid of circRNA_0026344, was transfected into HBE cells, the CSE-induced up-regulation of miR-21 levels was reversed. In MRC-5 cells, HBE-secreted exosomal miR-21 decreased levels of Smad7 and activated the TGF-β1/Smad3 pathway. By using the Targetscan database, the presence of species-conserved miR-21 binding sites in the Smad7 3'UTR region were predicted. We verified, by use of a luciferase reporter gene, that miR-21 bound to the 3'UTR region of Smad7 mRNA to inhibit its transcription. In conclusion, the results reveal that, in CS-induced pulmonary fibrosis, circRNA_0026344, via exosomal miR-21 regulation of Smad7, is involved in aberrant cross-talk of epithelium-fibroblasts. These results will be useful for the discovery of early biomarkers and for providing therapeutic targets for smoking-induced pulmonary fibrosis.

Keywords: Cigarette smoke; Exosomes; Pulmonary fibrosis; circRNA_0026344; miR-21.