Thle 2

All cell lines were purchased from ATCC (Manassas, VA) and grown in flasks in monolayers at 37°C with 5% CO₂. Four liver cancer cell lines were used, including HepG2/C3A (C3A) (derivative of HepG2, p53 WT), Hep3B2.1–7 (Hep3B, p53 null), PLC/PRF/5 (p53 dominant negative R249S mutation) , and Snu398 (p53 null), as well as four normal cell lines, THLE-2 (immortalized normal liver cells), hFOB1.19 (immortalized normal bone cells), IMR-90 (normal fetal lung cells), and BJ (normal foreskin fibroblasts). Cells were maintained in DMEM (C3A, Hep3B2.1–7, Snu398, PLC/PRF/5, and IMR-90), RPMI 1640 (Snu398), DMEM/F12 (hFOB1.19), or BEGM (THLE-2). Media was supplemented with 1% L-glutamine (Corning), 1% penicillin-streptomycin (Gibco), and 10% FBS (Atlanta Biologicals). THLE-2 cell media was also supplemented with 5 ng/mL epidermal growth factor (Corning) and 70 ng/mL phosphoethanolamine (Sigma-Aldrich), and the gentamycin/amphotericin and epinephrine provided with the BEGM media kit (Lonza, Walkersville, MD) were discarded as per ATCC instructions. THLE-2 cells were grown in flasks coated in fibronectin (Sigma-Aldrich), bovine serum albumin (Sigma-Aldrich), and bovine collagen (Advanced Biomatrix) as per ATCC instructions. All experiments were conducted between cell passages 5 and 20.

Human normal hepatocytes (THLE-2) and the human HCC cell line Huh7 were obtained from American Tissue Culture Collection. THLE-2 and Huh7 cells were cultured in DMEM (Corning, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and incubated at 37˚C in a 5% CO₂ incubator. A DDP-resistant cell line, Huh7/DDP, was established by exposing parental Huh7cells to increasing concentrations of DDP for 1 year, as previously described (26 (link)). Huh7 or Huh7/DDP cells were transfected with 100 nM mimic control (5'-UUCUCCGAACGUGUCACGUTT-3'; Shanghai GenePharma Co., Ltd.), 100 nM miR-361-5p mimic (5'-ACGCCUGGAGAUUCUGAUAAUU-3'; Shanghai GenePharma Co., Ltd.), 1 µg control-plasmid (cat. no. sc-437275; Santa Cruz Biotechnology, Inc.), 1 µg MAP3K9-plasmid (cat. no. sc-410886-ACT; Santa Cruz Biotechnology, Inc.), 100 nM miR-361-5p mimic + 1 µg control-plasmid or 100 nM miR-361-5p mimic + 1 µg MAP3K9-plasmid at 37˚C for 48 h using Polyplus transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. 48 h after the transfection procedure was completed, subsequent experiments were performed.