TOP-FLASH reporter assay

TOP-FLASH reporter assay was applied to detect the activity of the Wnt-β-catenin signaling pathway, in which β-catenin enters into the nucleus to form a complex with the transcription factor T-cell factor (TCF)/lymphoid enhancer-binding (LEF) regulating the transcription of downstream gene expression. Cell densities of HOS cells and U2OS cells were separately adjusted to 1 × 10⁵ cells/ml using DMEM. Then these cells (500 μl per well) were inoculated into a 24-well plate, followed by transfection based on the instructions of the Lipofectamine 2000 kit (LF2000; Invitrogen, Carlsbad, CA, U.S.A.) when cells reached 80–90% confluence. Cells transfected with miR-27a inhibitor, miR-27a mimic, SFRP1 siRNA plasmids as well as their corresponding controls were separately co-transfected with TOP-flash plasmid and pRL-TK plasmid. After 48 h of transfection, cells were added with 200 μl prepared passive lysis buffer (PLB, 1:4) and centrifuged for 3 min (12000 rpm, 4°C). Then supernatant (10 μl) was obtained, followed by centrifugation for 1 min (12000 rpm, 4°C). The luciferase assay reagent II (LAR II) and Stop & Glo reagent were used to detect Luc (Firefly luciferase activity) and Rel values. The transcriptional activity of TCF/LEF was visualized by the ratio of TOP-Flash Luc/Rel value (Ratio of TOP-Luc/Rel).