The role of microRNAs in chronic lung allograft dysfunction

Abstract

Dysregulation of miRNAs has been implicated in obstructive airway diseases including TGF-β1 mediated Bronchiolitis Obliterans Syndrome (BOS), where TGF-β induced epithelial phenotype plasticity or Epithelial-Mesenchymal Transition (EMT) may also contribute to pathophysiology. This study investigated the role of miRNAs in TGF-β1 induced fibrosis and had two main aims: (1) Identification of key miRNAs crucial in TGF-β1 induced EMT and fibrosis, a key clinical feature of BOS and (2) investigating the role of selective miRNAs (miR-200b, miR-200c and miR-146a) in maintaining epithelial cell morphology during EMT using immortalised human bronchial epithelial cells (BEAS-2B cells) and human primary bronchial epithelial cells (PBECs). Initially, NanoString® nCounter miRNA assay was used to profile miRNAs in control versus TGF-β1 stimulated BEAS-2B cells. MiR-200b and miR-200c were downregulated while miR-146a was upregulated post TGF-β1 treatment compared to control BEAS-2B cells. BEAS-2B cells and PBECs were transfected with miR-200b and miR-200c mimics that maintained the expression of epithelial cell markers and downregulated mesenchymal cell markers in the presence of TGF-β1 at RNA and protein level. The same experiment when replicated in PBECs derived from lung allografts yielded similar results. Next, the effect of miR-200b/c mimics was evaluated in TGF-β1 pre-treated cells. MiR-200b and miR-200c mimics reversed established TGF-β1 driven EMT in BEAS-2B cells. Furthermore, miRNA target studies were performed using computational tools, and a luciferase assay validated ZNF532 and ZEB2 as direct targets of miR-200b and miR-200c. Importantly, in situ hybridization revealed miR-200b-3p expression in the healthy lung epithelium. Cells transfected with miR-146a did not show any significant changes in EMT marker expression indicating some specificity to the miR-200b/c data. In conclusion, these investigations showed that miR-200b and miR-200c protect airway epithelial cells from EMT. Use of miR-200b/c mimics may therefore represent a novel therapeutic modulator of EMT associated with BOS.