Hibec

Four cell lines of cholangiocarcinoma (CCLP1, HuCCT1, RBE, 9810), the bile duct epithelial cell line (HIBEC) cells were obtained from the American Type Culture Collection and were maintained in Dulbecco modified Eagle medium containing 10% (v/v) fetal bovine serum at 37 degree in 5% CO₂ condition. All cell lines were routinely tested to be negative for mycoplasma contamination. Transfection of siRNA and plasmids was performed using Lipofectamine 3000 Reagents (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions. The sequences of siRNAs against specific targets were listed in Table S1.

The human cholangiocarcinoma cell line FRH–0201, CCLP–1 and the human intrahepatic bile duct epithelial cell line HIBEC, which were all purchased from American Type Culture Collection(ATCC, USA), were cultured in RPMI–1640 (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, USA) at 37°C in a humidified chamber under 5% (v/v) CO2.
Forty matched samples of cancer-adjacent tissue and cholangiocarcinoma tissue were obtained from patients undergoing surgical resection at the Guangdong General Hospital, after written informed consent was obtained from all patients. The matched cancer- adjacent samples were obtained at least 5 cm away from the tumor site. This study was approved by the Research Ethics Committee of Guangdong General Hospital (The approval number was GDREC2015097H).

Human intrahepatic biliary epithelial cell (HIBEC) was obtained from American Type Culture Collection (ATCC) and CCA cell lines including HuCCT1, QBC939, and Huh-28 were preserved in our laboratory. HIBEC and all three CCA cell lines were cultured in RPMI-1640 with 10% FBS (Invitrogen Life Technologies, USA) and 1x penicillin/streptomycin (Gibco, USA) in a humidified air with 5% CO₂ at 37°C. Among all three CCA cell lines, HuCCT1 exhibited the highest expression level of miR-196-5p; therefore, the HuCCT1 cell line was selected for subsequent functional experiments. MiR-196-5p inhibitor (Inhi-miR-196-5p) as well as inhibitor-nc (Inhi-nc) was acquired from RiboBio (Shanghai, China) and both sequences are protected by patents. The final concentration of Inhi-miR-196-5p or Inhi-nc in the culture supernatant was 100 nM and all functional experiments were performed 48 hours post-transfection. A HAND1 plasmid, which was applied to clone the whole HAND1 sequence, was purchased from GenePharma (Shanghai, China). The sequence details are provided in Table 2. Lipofectamine 3000 (Invitrogen Life Technologies, USA) was used for transfection according to the manufacturer's protocol.