microRNA regulation of WISP1 in pulmonary fibrosis: an in silico approach

Idiopathic pulmonary fibrosis (IPF) is the most common and devastating disorder of the lung interstitium. It is characterized by repetitive injury of the alveolar epithelium, fibroblast activation, and increased extracellular matrix production. MicroRNAs (miRNAs) are short single stranded nucleotides regulating protein expression. Differenzial expression of miRNAs has been described to act on pro-fibrotic target genes in lung fibrosis. Recently, we identified WNT1-inducible signaling pathway protein 1 (WISP1) as a novel alveolar epithelial cell-derived mediator in IPF. Here, we aim to investigate whether WISP1 expression is regulated by microRNAs in IPF.

Two miRNA array profiles from human IPF samples (9 diseased and 6 controls, 10 diseased and 10 controls) were analyzed for significant miRNA changes. Additionally, one murine miRNA dataset containing different time points from the murine bleomycine model was considered. In silico binding prediction of miRNAs to WISP1 3'-UTR was performed considering miRNA expression profiles from experimental and human idiopathic fibrosis tissue specimen. In vitro binding assays of miRNAs to WISP1 3'-UTR were conducted.

We analysed three published miRNA expression profiles, two human IPF and one murine bleomycin dataset. Three microRNAs, miR-30a, miR-30d and miR-92a, are significantly down regulated in all three studies (Log2 Fold Change: miR-30a, miR 30d and 92a are between -1,3 to -0,17, -1,76 to -0,23 and -1,15 to -0,03, respectively.) In silico binding analysis revealed that these three miRNAs bind to the WISP1 3'-UTR. Binding sites for miR-30 family in the 4kbp WISP1 3'UTR are at position 1183, 1194, 1811, 2657, 2982, 3525 and for miR-92a at 1600, 1947, 2583, 3027, 3369. For further analysis, we cloned WISP1 3'-UTR into a dual luciferase reporter plasmid. We performed reporter assays using the human alveolar epithelial cell line A549 and miR-mimics to confirm binding of these miRNAs to the WISP1 3'UTR.

The microRNAs-30a, -30d, and -92a are down regulated in IPF tissue and experimental fibrosis and may be involved in WISP1 upregulation and its subsequent pro-fibrotic action in the lung. In silico prediction revealed that miRNAs can bind to WISP1 3'-UTR and therefore, regulate WISP1 expression. In vitro reporter assays are performed to confirm the prediction analysis. Targeting specific miRNAs may represent a novel therapeutic strategy in IPF.