Dual-luciferase reporter assay
A luciferase reporter assay was carried out using a Dual-Luciferase Reporter Assay System (Promega, WI, U.S.A.). A fragment of the Wnt3a 3′UTR containing the predicted binding site for miR-195-5p or the respective binding site of the mutant-type (mut) 3′UTR was inserted into the pmirGLO vector. All the constructs were verified by DNA sequencing. The vector containing wild-type (wt) or mut was transfected into the 293T cells with or without the miR-195-5p mimics. 24-48 hours after transfection, the luciferase activity was detected using the Dual-Luciferase Reporter Assay System and normalized to Renilla activity.