Human normal mammary epithelial cell line MCF-10A was cultured in DME/F12 medium (Gibco, USA) containing 5% horse serum, 10 μ g/ml insulin, 20 ng/ml EGF, 100 ng/ml choleratoxin, 0.5 μg/ml hydrocortisone, 100 IU/ml penicillin and 100 μg/ml streptomycin. Human breast cancer cell lines MCF-7 and T-47D were maintained in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 IU/ml penicillin and 100 μg/ml streptomycin. Other human breast cancer cell lines ZR-75-30 and SK-BR-3 were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin in humidified 5% CO2 at 37°C. MiR-20b mimics, miR-20b inhibitor, antagomir-20b and their negative control were synthesized by Ribobio (RiboBio Co. Ltd., Guangzhou, China) [29 (link)].
The miR-20b gain-of-function study was performed using MiR-20b mimics (50 nM) and its negative control (50 nM) on the breast cancer cell lines. The loss-of-function study was performed with miR-20b inhibitor (100 nM) and its negative control (100 nM) on the cell lines. The cells were transfected using Lipofectamine RNAiMAX reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions.
The miR-20b gain-of-function study was performed using MiR-20b mimics (50 nM) and its negative control (50 nM) on the breast cancer cell lines. The loss-of-function study was performed with miR-20b inhibitor (100 nM) and its negative control (100 nM) on the cell lines. The cells were transfected using Lipofectamine RNAiMAX reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions.
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