Luciferase assay system

The interaction between HOXC10 and Slug in A375 and A2058 cells was examined by luciferase reporter assay. The vector pGL3-basic containing slug promoter (pGL3-basic-slug promoter) was transfected into cells using Lip2000 Reagent. Cells were co-transfected with pGL3-basic-slug promoter and different concentration of pcDNA3.1-HOXC10 (0, 50, 100, 200 ng). The activities of luciferase were measured using Dual luciferase assay kit (Promega, Madison, USA) on luciferase assay system (Ambion, Austin, TX, USA).

IRF4 containing the predicted miR-320c, miR-27a-3p or miR-30a-5p binding sites were cloned into pGL3-IRF4-Wt (wild type), pGL3-IRF4-Mut (mutant type) vectors (RIBOBIO) respectively. The miR-320c mimic, miR-27a-3p mimic, miR-30a-5p mimic and the corresponding mimic NC were synthesized by RIBOBIO. The Wt (Mut) 3′untranslated region (UTR) of IRF4 vector and miR-320c mimic, miR-27a-3p mimic, miR-30a-5p mimic or the corresponding mimic NC were co-transfected into 293 cells by using Lipofectamine 2000 Transfection Reagent (Invitrogen). pRL-TK vector was transfected into 293 cells as a reference for normalization. After 48 h of transfection, Dual luciferase assay kit (Promega, Madison, USA) was used to measure the activities of firefly and renilla luciferase on luciferase assay system (Ambion, Austin, TX, USA). The relative Rluc/Luc ratio was calculated to analyze the relationship among IRF4, miR-320c, miR-27a-3p and miR-30a-5p.

sPLA2-IIA containing the predicted miR-122-5p binding site were cloned into pGL3-sPLA2-IIA-Wt (wild-type) and pGL3-sPLA2-IIA-Mut (mutant type) (RiboBio Co., Ltd. Guangzhou, China), respectively. The Wt or Mut 3′-UTR of sPLA2-IIA vector and miR-122-5p mimic or its NC were co-transfected into 293 T cells utilizing Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific Inc.). The luciferase activity within the cells was measured 48 h post-transfection utilizing the luciferase assay system (Ambion, Austin, TX, United States).