Human lung cancer cell lines, including H522, H460, SK-MES-1, and A549, and a human bronchial epithelial cell line BEAS-2B, were from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cell lines H522, H460, and A549 were cultivated in RPMI-1640 medium (Gibco; Thermo Fisher Scientific), while SK-MES-1 cells were in minimal medium (Gibco). Both media contained 10% FBS (Gibco) and 1% penicillin-streptomycin mixture (Gibco). BEAS-2B cells were exposed to bronchial epithelial growth medium (Lonza/Clonetics, Walkersville, MD, USA) containing 0.5 ng/mL epidermal growth factor, 500 ng/mL hydrocortisone, 0.035 ng/mL bovine pituitary extract, 500 mM ethanolamine, 500 ng ethanolamine phosphate, 0.01 mg/mL epinephrine and 0.1 g/mL retinoic acid. All cells were maintained at 37°C in the presence of 5% CO2.

The synthesized miR-133a mimic and negative control (NC) mimic (GenePharma, Shanghai, China) were resuspended in 20 µM diethyl pyrocarbonate water. Briefly, NC mimic, miR-133a mimic, and Lipofectamine 3000 (Invitrogen) were each diluted with 100 µL serum-free medium. The diluted Lipofectamine 3000 was added to the diluted NC mimic or miR-133a mimic, respectively, and incubated at ambient temperature for 30 min. The mixture was subsequently added to cell suspensions of A549 and H1299. Subsequently, A549 and H1299 cells stably overexpressing miR-133a were infected with lentiviral vectors of overexpression (oe)-NC, oe-PTBP1 or oe-KDM5C for 2 days. The expression of miR-133a, PTBP1, and KDM5C in cells were verified using RT-qPCR or Western blot to confirm successful infection/transfection in the cells.

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