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.