Supplementary Material for: MicroRNA-200a Inhibits Transforming Growth Factor β1-Induced Proximal Tubular Epithelial-Mesenchymal Transition by Targeting β-Catenin

Background: The epithelial-mesenchymal transition (EMT) is a crucial event in the development of renal interstitial fibrosis (RIF). A growing body of evidence indicates that β-catenin plays an important role in various types of fibrosis. Although members of the microRNA (miRNA)-200 family have been suggested to suppress EMT in cancer and fibrosis, the function of miRNA-200a in regulating the progression of RIF is unknown. We speculate that miRNA-200a may hinder this progression through the suppression of β-catenin. Methods: Transforming growth factor β1 (TGF β1) was used to induce EMT of proximal tubule epithelial (HK-2) cells in vitro, quantitative real time polymerase chain reaction (qPCR) and Western Blot analysis measured the miRNA-200a and β-catenin expression. qPCR, Western Blot analysis, Migration Assay and cell immunofluorescence were applied to detect the influence of up- and downregulated miRNA-200a expression and β-catenin siRNA on β-catenin and EMT. Dual luciferase report plasmid (CTNNB1 UTR WT/MT) verifies the target relationship between miRNA-200a and CTNNB1 (β-catenin gene). Results: miRNA-200a is downregulated and β-catenin is upregulated during TGF β1-induced EMT. Upregulation of miRNA-200a inhibits β-catenin and attenuates TGF β1-induced EMT and cell migration, while its downregulation increases β-catenin and induces EMT and HK-2 cell migration. Further, knocking down β-catenin suppressed the EMT induced by miRNA-200a downregulation. miRNA-200a directly targets for CTNNB1. Conclusions: miRNA-200a inhibits TGF β1-induced EMT by directly targeting β-catenin in proximal tubule epithelial cells.