Sk mes 1

Human normal lung epithelial cells (BEAS−2B cells), lung squamous cell carcinoma cells (SK-MES−1, NCI-H520, and Calu−1 cells), lung adenocarcinoma (LUAD) cells (A549, H1975, and PC-9 cells), and 293T cells were purchased from ATCC (VA, USA). All cells were cultured in DMEM (Gibco, MD, USA) containing 10% FBS (Gibco) with 5% CO₂ at 37°C. A549/DDP and H1975/DDP cells were constructed as previously described^{22 (link)}. Cells were treated with cisplatin for 2 d as a cycle. After completing three cycles of stimulation with the same concentration of cisplatin, the dose was increased from 2 μg/ml to 50 μg/ml.

Human NSCLCs and matched normal tissues were obtained from 30 patients in our department. Informed written consent was obtained from all patients, and this study was approved by the Medical Ethics Committee at Zhongnan Hospital. All tissue samples were stored in liquid nitrogen until RNA extraction and western blotting. Clinicopathologic information is summarized in Table 1. The NSCLC cell lines A549, SK-MES-1, H460 and H520 and the normal lung bronchus epithelial cell line 16HBE were obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. All cells were maintained in a humidified, 5% CO₂ atmosphere at 37 °C. After the NSCLC cells reached 80% confluence, they were transfected with miR-152 or control mimics using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.