Mhcc97 h

Normal human HL-7702 hepatocytes and HepG2, HUH-7, MHCC-97H, and PLC HCC cell lines were purchased from the BeNa Culture Collection (Beijing, China). All cells were grown in Dulbecco’s modified Eagle medium (DMEM; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.), 100 U/mL streptomycin, and 100 U/mL penicillin at 37°C in an atmosphere of 95% air and 5% CO₂.
Cell transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. HepG2 cells grown to 70% confluency were transfected with scramble control (sh-NC), short hairpin RNA (shRNA) against miR-133a-3p (sh-miR-133a-3p), mimic control (mimic-NC), or miR-133a-3p mimics (miR-133a-3p). For luciferase reporter assay, HepG2 cells were co-transfected with pGL3 reporter constructs containing the wild-type 3ʹ-UTR of SPCOD1 (SPOCD1-WT) or a 3ʹ-UTR with a mutant miR-133a-3p-binding site (SPOCD1-Mut), and mimic-NC or miR-133a-3p. The above plasmids were purchased from Shanghai Genechem Company (Shanghai, China). The medium was replaced with fresh culture medium 6 hours after transfection, and the cells were cultured until the next experiment.

The liver cancer cell lines Huh7, BEL-7402, HepG2, HCCLM3, and MHCC97H, as well as the normal hepatocyte cell line LO2, were obtained from the BeNa Culture Collection (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco) was used to incubate the cells. Then, these cells were cultured in a humidified atmosphere with 37°C and 5% CO₂.

Human umbilical vein endothelial cells (HUVECs), HCC cell lines (MHCC97-H, HCCLM3 and Huh7) and normal human liver cell line THLE-2 were provided via Bena Culture Collection (Beijing, China). DMEM (Sigma, St. Louis, MO, USA) with 10% fetal bovine serum (Biosun, Shanghai, China) as well as 1% penicillin–streptomycin (Sigma) was applied to cell culture. The cells were maintained at 37 °C in 5% CO₂, and medium was changed every 3 days. To block the release of exosomes, cells were incubated with 10 μM of SW4869 (Sigma).