Hepg2

The human hepatoma cell lines HepG2, Huh7, and HepG2.2.15 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HepAD38 was provided by Professor Zhi Li, College of Life Sciences, Shaanxi Normal University.Ethics approval for the work was granted by the Ethics Committee of The Second Xiangya Hospital.Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, Gibco, New York, USA) containing 100 U/mL penicillin and streptomycin (Cat. No. ST488, Beyotime, Beijing, China) at 37°C in a humidified atmosphere with 5% CO₂. Cells were plated into 6-well plates at a density of 1×10⁶ cells/well. After 24 hours, a concentration gradient of the HBV-miR-3 agomir or of the negative control (NC) RNA (artificially synthesized by GenePharma, Shanghai, China) was transfected into HepG2 cells and into HepAD38 cells. Transfection was performed using GP-siRNA-Mate Plus (GenePharma, Shanghai, China) according to the manufacturer’s instructions. The growth medium was changed after 8 hours. Transfected cells were harvested at 48 hours, and total cellular RNA and protein were isolated for RT-qPCR and Western blot analyses. All transfections were performed in triplicate. The blank group refers to cells that were not transfected with any RNA.

The human liver cancer cell lines Huh7, HepG2, and HepG2.2.15 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Huh7 and HepG2 cells were grown and maintained in DMEM, and HepG2.2.15 cells were maintained in Minimal Essential Medium-α (Gibco, BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco), 20 mM HEPES, 100 U/mL penicillin, and 100 µg/mL streptomycin. The Minimal Essential Medium-α cells were supplemented with 2 mM glutamine and 200 µg/mL G418. Primary human hepatocytes are purchased from Liver Biotechnology (Shenzhen, China) and cultured according to the manufacturer’s protocol. The cultures were incubated in a cell incubator in an atmosphere containing 5% CO₂ at 37°C. All transfections were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. The amount of transferred DNA for 24-well plates was 1 µg, for 6-well plates, 3 µg, and for 100 mL culture flasks, 10 µg. Primary human hepatocytes were infected with the concentrated culture supernatant of HepG2.2.15 cells as described.^{30 (link)}

Human HCC cells (HepG2, Hep3B, Li7, Huh-7, HCC-LM3 and Hep-G2) were originally purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Normal liver cells (L02) were originally purchased from Fuxiang Biotechnology Company (Shanghai, China). L02, Huh-7, HCC-LM3, and Hep-G2 cell lines were cultured in DMEM medium plus 10% fetal bovine serum (FBS). Hep-3B cell lines were cultured in MEM medium plus 10% FBS, 1% non-essential amino acids, and 1% sodium pyruvate. Li-7 cell lines were cultured in MEM medium plus 10% FBS. All cell lines maintained in a 5% CO2 incubator at 37 °C [26 (link)].