Luciferase reporter vector

The human HCC HepG2 and SMMC-7721 cell lines and immortalized normal liver epithelial THLE-3 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were maintained under the conditions stated by the supplier.
Mature miR-592 mimics, miR mimics negative control (NC) and luciferase reporter vector were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). Prior to cell transfection for 1 h, cell culture medium was replaced with Dulbecco's modified Eagle's medium (DMEM) medium without antibiotics and fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc. Waltham, MA, USA). Cells were transfected with miR-592 mimics, NC, or co-transfected with the luciferase reporter vector using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequent to transfection for 4–6 h, cells were washed with PBS and cultured at 37°C with 5% CO₂ according to the manufacturer's conditions in DMEM without antibiotics.

Luciferase reporter vector (Shanghai GenePharma Co., Ltd.) with the LBX2-AS1 promoter -2,500 kb ~ +245 bp full sequence and LBX2-AS1 promoter deletion plasmids were transfected into 293T cells (ATCC) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The fluorescence signal was monitored after 24 h of transfection. LBX2-AS1 wild-type (LBX2-AS1-wt) and mutant (LBX2-AS1-mut), S100A11 wild-type (S100A11-wt) and mutant (S100A11-mut) sequences were inserted into the pmirGLO vector (Promega Corporation). Mutant sequences were generated using a Hieff Mut™ Site-Directed Mutagenesis kit (cat. no. 11003ES10; Shanghai Yeasen Biotechnology Co., Ltd.). miR-491-5p mimics/controls were co-transfected with LBX2-AS1-wt/LBX2-AS1-mut or S100A11-wt/S100A11-mut using Lipofectamine 2000. After 48 h, the cells were collected and luciferase activity was determined using a Dual-Luciferase Reporter Assay System (Beyotime Institute of Biotechnology). These results were standardized in line with Renilla luciferase activity.