Mir 106b mimics

CCA cells were seeded in 96-well plates (4×10³ cells per well) and at 37°C in an incubator containing 5% CO₂. Cells were treated with 40 μM 5-FU, 40 μM 5-FU plus miR-106b mimics (Genepharma, Suzhou, China). Cell viability was tested by using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) assay at 0 h, 12 h, 24 h after treatment. Briefly, cells were incubated with MTT at a final concentration of 0.5 mg/mL for 4 h. The supernatant was discarded, and the precipitated formazan was dissolved in dimethyl sulfoxide. Absorbance was measured at 490 nm with micro-plate reader (Molecular Devices, i3).

The mimics control, miR-106b mimics, inhibitor control, anti-miR-106b inhibitor, scrambled siRNA control, Tp53inp1 siRNA, and Cdkn1a siRNA were purchased from GenePharma (GenePharma Co., Ltd., Shanghai). Transfection of miRNA mimics/inhibitor or siRNAs was performed using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instruction.

The mimics control with scrambled sequence, miR-106a mimics, miR-106b mimics, miR-124 mimics, inhibitor control with scrambled sequence, miR-106b inhibitor, miR-124 inhibitor, miR-22 inhibitor, and miR-29b inhibitor were purchased from GenePharma (GenePharma Co., Ltd., Shanghai, China). Transfection of miRNA mimics/inhibitor or their corresponding control (20 nM) was performed using the HiPerFect Transfection Reagent (Qiagen) according to the manufacturer’s instruction.