Dual luciferase activity system

Wild-type (wt) sequence of CASC9 or S100A14 contains a binding region for miR-335-3p was inserted into psi-CHECK-2 (Promega. Madison, WI, USA) to generate wt-CASC9 or wt-S100A14. Mutant luciferase vectors (mt-CASC9 or mt-S100A14) were constructed using site-direct mutagenesis kit (Takara) to lose miR-335-3p binding sites. Cells were co-transfected with luciferase vectors and miRNAs using Lipofectamine 2000. 48 hrs later, dual-luciferase activity system (Promega) was utilized to measure relative luciferase activity using Renilla luciferase as internal control.

FHL3 was selected for analyses after TargetScan analysis as it ranks top among all predicted targets for miR-663a and was found to play tumor suppressive role in cancers. Wild-type (wt) or mutant (mt) 3’-untraslated region sequence of FHL3 contains binding region for miR-663a was inserted into pGL (Promega, Madison, WI, USA) to generate FHL3-wt or FHL3-mt luciferase constructs. Cells were co-transfected with luciferase constructs and miRNAs using Lipofectamine 2000. After 48 h, relative luciferase activity was measured using dual-luciferase activity system (Promega) in accordance with the manufacturer’s protocols.

Chemically synthesized wild-type (WT) 3′-untranslated region sequence of DSCAM-AS1 was inserted into a pMIR-reporter (Promega Corporation) to generate a WT-DSCAM-AS1 construct. A site-direct mutagenesis kit (Takara Biotechnology Co., Ltd.) was used to generate MT-DSCAM-AS1 construct from WT-DSCAM-AS1. WT-DSCAM-AS1 or MT-DSCAM-AS1 and the aforementioned miRNAs were co-transfected into EC cells using Lipofectamine^® 2000 according to the manufacturer's instructions. After 48 h, cells were collected to measure relative luciferase activity using the Dual-luciferase activity system (Promega Corporation) with Renilla luciferase activity used as the internal control.