Mirna mimic inhibitor

BEL7402, SMMC7721 and HepG2 hepatocarcinoma cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum. Anchorage-deprived cell model was constructed as previously described9 (link)10 (link). Human HEK293 cells were obtained from American Type Culture Collection, and were cultured at 37°C with 5% CO₂ in DMEM supplemented with 10% FCS. The transient transfections for cell models were performed with Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, USA). For miRNA transfection experiments, exponentially growing HCCs were plated in 6-well plates at a density of 2 × 10⁵ cells/well and were grown overnight to 80% confluence. An miRNA mimic/inhibitor or a nonsense control (GenePharma Co., Shanghai, China) was put to the culture medium at a final concentration of 100 nM according to the manufacturer's recommendations. For the ICAT/CTNNBIP1 inhibition and overexpression studies, siRNA (GenePharma Co., Shanghai, China) against ICAT or HA-tagged ICAT plasmid (a kind gift from Dr. Tetsu Akiyama from University of Tokyo) was introduced to the HCC cells at the final concentration of 50 nM according to the protocol.

miR-101 mimics, anti-miR-101 and their respective negative controls (miRNA mimic/inhibitor) were synthesized by Shanghai GenePharma Co., Ltd. The full length of Rap1b was subcloned into pcDNA3.1 to overexpress Rap1b, with empty pcDNA3.1 vector serving as the control. Transfection of the cells with the miR-101 mimics (10 nM), anti-miR-101 (10 nM), miRNA mimic/inhibitor (10 nM) and vectors (10 nM) were performed using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.). Co-transfection of the cells with miR-101 mimic and Rap1b was performed using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), the subsequent experiments were performed at 48 h post-transfection. The sequences of oligonucleotides used were as follows: miR-101 mimics, 5′-UACAGUACUGUGAUAACUGAA-3′; miRNA mimics, 5′-UUUGUACUACACAAAAGUACUG-3′; anti-miR-101, 5′-UUCAGUUAUCACAGUACUGUA-3′ and miRNA inhibitor control, 5′-UCACAACCUCCUAGAAAGAGUAGA-3′.