Anti con

Chemically synthesized miRNA mimics or inhibitors to overexpress or inhibit miR-223 and each unrelated negative control (miR-Con or anti-Con), were purchased from Ribobio (Guangzhou, China). Cells at 70% confluence after overnight culture on petri dishes were transfected with miR-223 mimic, mimic control (miR-Con) (50 nmol/L), or miR-223 inhibitors (anti-223), inhibitor control (anti-Con) (100 nmol/L) by Lipofectamine 2000 (Invitrogen). After 6 h transfection, medium was changed, and the cells were cultured for 24 h and then exposed to either hypoxic or normoxic conditions for 24 h or 48 h.

Anti-con, anti-miR-148a, Con-mimic, and miR-148a-mimic were synthesized by RiBoBio Co. Cells were transiently transfected using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA) for 12 h, according to the manufacturer's protocol. For gene recovery assay, after MHCC97H cells were transfected by anti-miR-148a for 12 h, they were cultured in fresh DMEM medium supplemented with 10% FBS (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco) for another 24 h, followed by transfected with Con-mimic or miR-148a-mimic for 12 h.

The siRNAs targeting IL-6R or STAT3 were purchased from RiBoBio Co (Guangzhou, China) and transfected into MGC803 cells using the Lipofectamine^® 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Con-mimic, miR-124-mimic, anti-con, and anti-miR-124 were also synthesized by RiBoBio. Briefly, cells were seeded into six-well plates at a density of 1 × 10⁵ cells per well for 24 h and then were transfected with 50 nM anti-miR-124 or 20 nM miR-124-mimic for 12 h. After transfections, cells were cultured in fresh RPMI-1640 medium supplemented with 10% FBS (Gibco-BRL) for another 24 h before using for other experiments.