The interaction between miR-143 and HMGA2 was analyzed by a dual-luciferase reporter assay [23 (link)]. The potential binding site of miR-143 on HMGA2 was predicted by Targetscan and microrna.org databases. Wild-type binding sites and flanking sequence (~200bp) were cloned into the pMIR-REPORT Luciferase vector at HindIII and SpeI sites (Ambion; Thermo Fisher Scientific). To generate the pMIR-REPORT Luciferase vector carrying the miR-143 binding site mutant sequence, Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific) was used to introduce several point mutations into the HMGA2 3’-UTR. The correct sequences of HMGA2-WT and HMGA2-Mut were confirmed by DNA sequencing.
After that, we inoculated TPC-1 cells into the 6-well plates, followed by transfection using Lipofectamine 2000 (Invitrogen) by specific vectors for 48 h. Afterward, we adopted a Dual-Luciferase-reporter 1000 detection system (Promega, Madison, WI, USA) to detect luciferase activities normalized to Renilla.
After that, we inoculated TPC-1 cells into the 6-well plates, followed by transfection using Lipofectamine 2000 (Invitrogen) by specific vectors for 48 h. Afterward, we adopted a Dual-Luciferase-reporter 1000 detection system (Promega, Madison, WI, USA) to detect luciferase activities normalized to Renilla.
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