The role of TGF-β1 in mediating extracellular matrix remodeling during the pathogenesis of lung fibrosis has been well defined. Dysregulated expression of miRNA is increasingly implicated in various pathological processes and may play an important role in progressive loss of lung function. This study was designed to evaluate the expression of miRNAs in TGF-β1 treated BEAS-2B cells (immortalized bronchial epithelial cells) and further investigate the role of miR-200b in modulating TGF-β1 induced epithelial-mesenchymal transition (EMT) in lung fibrosis.

The differential expression of miRNAs were assayed using miRNA Expression Assay kit (Nanostring, Seattle) in untreated and 1, 4 and 24 hrs TGF-β1 treated BEAS-2B cells. MiR-200b expression was validated using quantitative real time PCR (qRT-PCR). BEAS-2B and PBECs (primary bronchial epithelial cells) were transfected with miR-200b mimics to study expression of EMT markers at mRNA and protein level using qRT-PCR, immunofluorescence and western blot respectively. MiR-200b targets were identified and validated using multiple computational tools and qRT-PCR respectively. In-situ hybridization allowed detection of miR-200b in tissues samples at cellular level.

NanoString^TM allowed identification of differentially expressed miRNAs following TGF-β1 treatment. BEAS-2B cells and PBECs treated with TGF-β1 showed a significant decrease in E-Cadherin (p≤0.001) and an increase in Fibronectin (p≤0.001) expression as compared to untreated and miR-200b transfected cells alone. MiR-200b transfected cells following TGF-β1 treatment restored the epithelial phenotype by significantly upregulating E-Cadherin (p≤0.05) and downregulating Fibronectin (p≤0.001). Immunofluorescence and western blot also showed a similar trend of EMT marker expression in both the cell types. MiR-200b significantly reduced the expression of its targets ZNF532 (p≤0.01) and ZEB2 (p≤0.001) in BEAS-2B cells and ZNF532 (p≤0.01) in PBECs post TGF-β1 treatment. In-situ hybridization allowed localization of miR-200b in airway epithelium of normal human lung sections. Further studies will evaluate the expression of miR-200b in tissue sections from transplanted lung.

The findings suggest that miR-200b suppressed TGF-β1 induced EMT in BEAS-2B cells and PBECs. The outcomes from this study may offer new insights into mir-200b regulation in fibrosis and have potential for therapeutic application in progressive airway diseases.

CITATION INFORMATION: Ladak S, Ward C, Ali S. MiRNA-200b Inhibits Epithelial-Mesenchymal Transition in TGF-β1 Induced Human Bronchial Epithelial Cells. Am J Transplant. 2016;16 (suppl 3).

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