Dual glo luciferase analysis system

The sequence of armadillo-repeat-containing protein 8 (ARMC8) containing wild-type miR-142-5p binding site was cloned into pGL3 reporter gene vector (Promega, Madison, Wisconsin, USA) to construct wild-type ARMC8. Meanwhile, the sequence of ARMC8 containing mutant-type miR-142-5p binding site was cloned into pGL3 to construct mutant-type ARMC8. Then, miR-142-5p mimic or mimic NC was co-transfected with the constructed reporter plasmid into U2OS or HOS cells. After 48 h incubation, the culture medium was discarded and cells were harvested. The harvested cells were lysed to produce cell lysate. Luciferase activity was measured using the Dual-Glo® luciferase analysis system (Promega).

The possible binding site of miR-34b-5p with SNHG3 or HOXC6 was obtained from the Starbase database. Next, the sequence of SNHG3 or HOXC6 containing wild-type (WT) or mutant-type (MUT) miR-34b-5p binding site was synthesized (RiboBio) and cloned into the pGL3 reporter gene vector (Promega Corporation, Madison, Wisconsin, USA). The vectors were co-transfected with miR-34b-5p mimic or mimic NC (GenePharma) into HCT116 or SW480 cells using the Lipofectamine 3000 reagent (Invitrogen) and incubated for 48 h. Afterwards, luciferase activity was measured using the Dual-Glo® Luciferase Analysis System (Promega).