Hibepic

The human normal intrahepatic biliary epithelial cell line (HIBEpiC) was purchased from the BeNa Culture Collection and cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin. 293 T cells were obtained from the Cell Bank of the Chinese Academy of Science and cultured in Dulbecco’s modified Eagle medium (DMEM). The two cell lines were maintained in a humidified incubator with an atmosphere of 5% CO₂ at 37 °C. Subsequently, the HIBEpiC cells were induced into EMT using a conditioned medium containing TGF-β1 (10 ng/ml) for 48 h, as previously described [19 (link)].
The oligonucleotides of the miR-29c mimics, miR-29c inhibitor and their corresponding negative controls (mimics NC and inhibitor NC, respectively) were synthesized at Genepharma Co., Ltd. For cell transfection, HIBEpiC cells were seeded in six-well plates and grown to 80% confluence, followed by transfection with miR-29c inhibitor using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer’s protocol. For DNMT3A and DNMT3B suppression, HIBEpiC cells transfected with miR-29c inhibitor were treated with DNMT3A/DNMT3B inhibitor SGI-1027 (Selleckchem) for 36 h.

Three cell lines of cholangiocarcinoma (RBE, QBC939, and FRH0201), the bile duct epithelial cell line (HIBEpiC), and the human umbilical vein endothelial cell line (HUVECs) were bought from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Mycoplasma contamination testing was performed to confirm the negative infection of mycoplasma contamination. The RBE, QBC939, and FRH0201 cells were cultured in RPMI 1640 culture medium (Gibco) containing 10% fetal bovine serum (FBS). The HIBEpiC cells were cultured in a DMEM culture medium (Gibco) containing 10% FBS. The HUVECs cells were cultured in Media 199 culture medium (Gibco) containing 10% FBS. All cells were grown in a humidified 5% carbon dioxide (CO₂) at 37 °C.

The human intrahepatic bile duct epithelial cells (HIBEpiCs) were purchased from ICell Bioscience Inc. (Shanghai, China). The human hepatocellular carcinoma cell line MHCC-97h and human mononuclear cell line THP-1 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The HIBEpiC and THP-1 were cultured in RPMI-1640 medium (Gibco, Grand Island, USA), and MHCC-97h was cultured in DMEM medium (Gibco, Grand Island, USA), all media containing 10% fetal bovine serum (Gibco, Grand Island, USA) and 1% penicillin/streptomycin (Beyotime, Shanghai, China). All three cell lines were grown in an incubator (37°C, 5% CO₂).
Cobalt chloride (CoCl₂, Sigma-Aldrich, USA) was used to simulate the hypoxia process of HIBEpiCs. The HIBEpiCs were divided into four groups: the control group (CON), the IL-22 (10 ng/ml) (Recombinant human IL-22, Absin, Shanghai, China) treatment group, the CoCl₂ (150 μM) treatment group, and the CoCl₂ (150 μM)+IL-22 (10 ng/ml) treatment group.