Synthetic mir 34a mimic

The human EHCC cell lines QBC939 and HuCCT1 used in this study were purchased from American Type Culture Collection (Manassas, USA) and the human IHCC cell line RBE and HCCC9810 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human intrahepatic biliary epithelial cells (HiBECs) were purchased from PriCells Biomedical Technology Co., Ltd. (Wuhan, China). All the cells were cultured according to the manufacturer’s instructions. A chemically modified antisense oligonucleotide and a synthetic miR-34a mimic (GenePharm Co. Ltd, China) were used to inhibit and increase miR-34a expression respectively. A scrambled oligonucleotide (GenePharm Co. Ltd, China) was used as a control. The transfections were performed using Lipofectamine TM 2000 transfection reagent (Invitrogen, USA) according to the manufacturer’s instructions. A mixture of Lipofectamine 2000 and RNA was added to CC cells, which were 70 % confluent, for 4–6 hrs, and the cells were then incubated for 24 hrs in fresh medium. After that, the cells were harvested using lysis buffer for luciferase assay. Total RNAs and protein were prepared 48 hrs after transfection and used for qRT-PCR or western blot analysis.

HCT8 and SW480 cells (1 × 10⁵ cells) were seeded in 6‐well plates in DMEM medium for 12 hours. Then, cells were transfected with 10 nmol/L mimic nontargeting control and the relevant miRNA mimic, and 10 nmol/L inhibitor nontargeting control and the relevant miRNA inhibitor through Oligofectamine transfection reagent. Next, HCT8 and SW480 cells (1 × 10⁵ cells) were plated in 6‐well plates and transfected with 5 nmol/L NEAT1 shRNA or shRNA negative control via 0.2% Lipofectamine 3000 in DMEM medium. The synthetic miR‐34a mimic, miR‐34a inhibitor and their negative control (miR‐control), specific shRNA targeting NEAT1 or scramble shRNA, and human HMGB1 overexpressing plasmid were obtained from GenePharma. After 5 hours, the transfection reagent was replaced with DMEM medium. Forty‐eight hours later, cells were collected for further experiments.