Mir 16 5p mimic

StarBase (http://starbase.sysu.edu.cn/) was utilized to predict the binding site of miR-16-5p to FOXK1. The mutant (mut) and wild-type (wt) sequences containing the binding sites of miR-16-5p to FOXK1 (mut-FOXK1 and wt-FOXK1) were designed and synthesized based on the predicted results and cloned into luciferase reporter vectors (pGL3-Basic). Thereafter, the designed sequences were co-transfected with miR-16-5p mimic (0, 150, 300 nM, GenePharma) into HEK293T cells, respectively. After that, cells received 20-min cell lysing with 100 μL cell lysis buffer on a shaking table at room temperature. Firefly luciferase activity or Renilla luciferase activity was measured after the exposure of cell suspension (50 μL) to luciferase reaction solution (50 μL, Promega, Madison, WI, USA) or Stop & Glo reagent (50 μL, Promega). The relative activity was calculated as the ratio of Firefly luciferase activity to Renilla luciferase activity. Renilla luciferase activity was regarded as an internal control. Three replicates were set for this test.

KHOS and 143B cells were cultured in six‐well plates (8 × 10⁵ cells per well) to 70%‐80% confluence before transfection. miR‐16‐5p mimic and negative control were synthesized by GenePharma. Lentivirus, the sh‐circUSP34 vector #1 and #2, was constructed targeting the specific head‐to‐tail junction site purchased from Hanbio Biotechnology Company. Sequences of mimic, inhibitor, and vectors are listed in Table 2. Lipofectamine 3000 (Invitrogen) and Polybrene (Hanbio Biotechnology Company) were applied to facilitate miR‐16‐5p mimic and sh‐circUSP34 complex formation, respectively.