Mhcc97 l

Human hepatocellular carcinoma cells MHCC97-L and HCCLM3 (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.), and incubated at 37°C in an atmosphere containing 5% CO₂. Media were supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). EA was purchased from Chengdu Must Bio-Technology Co., Ltd. and was dissolved in DMSO before being used to treat the hepatocellular carcinoma cell lines MHCC97-L and HCCLM3 at different concentrations (7, 14 and 28 µM) at 37°C for 24, 48 or 72 h, according to the subsequent experiment. The following inhibitors were used to treat the MHCC97-L and HCCLM3 cell lines for 30 min prior to EA treatment at 37°C: Z-VAD-FMK (20 µM; MedChemExpress LLC), necrostatin-1 (30 µM; MedChemExpress LLC), 3-methyladenine (3-MA; 2 mM; MedChemExpress LLC), N-acetylcysteine (NAC; 3 mM; MilliporeSigma) and PD98059 (20 µM; MedChemExpress LLC). DMSO was used to treat the control group. All experiments were performed independently and in triplicate.

Human normal liver cell line THLE3 and HCC cell lines (Hep3B, American Type Culture Collection, Rockville, Maryland, USA; MHCC-97L and Huh7, Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., Shanghai, China) were, respectively, seeded in Roswell Park Memorial Institute (RPMI)-1640 cell culture medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were cultured at 37°C with 95% saturated humidity.
The cells were seeded onto a 6-well plate 24 h before transfection, and the transfection process was performed in the light of Lipofectamine 2000 (11668-019, Invitrogen, New York, CA, USA) upon reaching 30–50% confluence. The cells were treated with miR-19a-3p inhibitor, miR-376c-3p inhibitor, miR-19a-3p inhibitor + miR-376c-3p inhibitor, overexpressed (oe)-SOX6, oe-SOX6 + miR-19a-3p mimic, or oe-SOX6 + miR-376c-3p mimic, as well as their corresponding negative controls (NCs). The plasmids were available from Shanghai GenePharma Co., Ltd. (Shanghai, China). The cells were cultured in an incubator at 37°C, and the complete medium was renewed 6 h later. The subsequent experiments were carried out in cells after a 48-h culture.