Cclp 1

Three ICC cell lines (CCLP-1, RBE and HuCCT-1) and a normal human intrahepatic biliary epithelial cell line (HIBEC) were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences, (Shanghai, China). Cells were cultivated according to the protocols from their supplier. All cell lines were grown in RPMI-1640 complete medium (Biological Industries, Kibbutz Beit-Haemek, Israel) supplemented with 10% fetal bovine serum (FBS; Moregate Biotech, Brisbane, Australia), and were cultured in an incubator of 37°C and 5% CO2.

Human cell lines, including the noncancerous cholangiocyte cell line HIBEC and the CCA cell lines RBE, CCLP1, HuCCT1 and HCCC-9810 were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). All cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and grown in humidified 5% CO₂ at 37°C. Short hairpin RNA (shRNA) targeting FLVCR1-AS1 (shFLVCR1-AS1) and the relative control shRNA (shNC) were obtained from Shanghai Genepharma Co., Ltd., (Shanghai, China). The sequence of shFLVCR1-AS1 was 5′-GGTAAGCAGTGGCTCCTCTAA-3′ and the sequence of shNC was 5′-AATTCTCCGAACGTGTCACGT-3′. The transfection was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in 5×10⁶ cells at a final concentration of 50 nM, according to the manufacturer's protocol. After transfection for 24 h, expression of FLVCR1-AS1 was validated by reverse transcription-quantitative PCR (RT-qPCR).