P tk renilla plasmid

The HOXA1 wild type (WT) 3′-UTR sequence was inserted into the downstream of the firefly luciferase gene in the psiCHECK™ vector (Promega), and the mutant (MT) plasmid was created by site directed mutagenesis. Then, the HOXA1 WT or MT 3′-UTR reporter plasmids, the p-TK Renilla plasmid (Promega), together with miR-99a mimics or inhibitor, were used to cotransfect CNE-2 and HONE-1 cells using Lipofectamine 2000 (Invitrogen). After 24 h, the cells were harvested and the Renilla and firefly luciferase activities were determined using the Dual Luciferase Reporter Assay System (Promega).

TargetScan, PicTar, and miRbase (http://www.mirbase.org/) were used to predict targets of miR-483-5p. In the luciferase reporter assay, the 3′-untranslated regions (UTRs) were designed according to bioinformatics software [18 (link)]. TargetScan, PicTar, and miRbase were used to design 3′-UTRs. MKNK1 sequence used was as follows: 5′-CGGAUGUCCUCUUUGAAACUCUCC-3′. The MKNK1 UTRs containing the predicted miR-483-5p binding sites and corresponding mutant sites were amplified, and were then inserted with the downstream luciferase gene in the psiCHECKTM vector (Promega Corporation). GHINK-1 cells were cultured in a 24-well plate at a density of 2.5×10⁵ cells/well 1 day prior to transfection. The final concentration (10 nM) of MKNK1 wild-type or mutant reporter plasmids, the p-TK Renilla plasmid (Promega Corporation), plus miR-483-5p mimics or controls, were used for co-transfection of GHINK-1 cells (4×10⁵ cells/well) using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocols. Luciferase activities were determined with a dual-luciferase assay kit (Promega Corp.) 48 h subsequent to transfection. The Renilla luciferase activity was measured as an internal control for each well.