Wild-type (WT) and mutant (MT) seed region sites of hsa-miR-1246 containing 3’UTR sequence of target genes were constructed and cloned into pMIR-REPORT-Luciferase vector (Invitrogen, Carlsbad, CA, USA) (Figure 3 B). Constructed insert sequences were verified by Sanger sequencing using BigDye Terminator v3.1 kit (Applied Biosystems, Foster City, CA, USA) and 3500 Series Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). A previously well-established workflow for dual luciferase assay using AGS cells was applied for this experiment [57 (link),58 (link)]. Cells were co-transfected with hsa-miR-1246 mimic and miR-Control, pMIR-REPORT-Luciferase-WT vector, pMIR-REPORT-Luciferase-MT vector, and pMIR-REPORT-ß-galactosidase control vector (Invitrogen, Carlsbad, CA, USA). Luciferase activity was detected after 48 h of incubation by the Dual-Light™ Luciferase & β-Galactosidase Reporter Gene Assay System (Invitrogen, Carlsbad, CA, USA) and normalized with ß-galactosidase activity using Tecan Genios Pro (Tecan, Männedorf, Switzerland). At least three independent experiments were performed.
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