Nci h1299

This study includes five pairs of lung adenocarcinoma (LUAD) tissues from Space Central Hospital. The ethical approval for this study was from Space Central Hospital (20,200,511-JJJHZ-01). Written informed consents were obtained from each patient. Four NSCLC cell lines (A549, NCI-H1299, HCC827, and NCI-H1975) and human bronchial epithelial cell line HBEC were obtained from Procell Life Science&Technology Co.,Ltd (Wuhan, China). A549 cells were cultured in DMEM (High glucose, Gibco, Grand Island, NY, USA, 11,965–092) with 10% FBS (Gibco) and 1% penicillin/streptomycin (P/S, Procell). NCI-H1299, HCC827, and NCI-H1975 cells were cultured in RPMI-1640 medium (Gibco) with 10% FBS (Gibco) and 1% penicillin/streptomycin (P/S, Procell). HBEC cells were cultured in the bronchial epithelial growth medium (BEGM, Lonza, CC-3170). All the cells were cultured at 37 °C in a 5% CO₂ incubator.

NCI-H1299 and NCI-H1650 cell lines were purchased from Procell Life Science and Technology Co (Wuhan, China), whereas HBE, A549, and HCC827 cell lines were gifted by the Key Laboratory of Environment and Genes Related to Diseases of Xi’an Jiaotong University. NCI-H1299, NCI-H1650, HCC827, and HBE cells were cultured in RPMI-1640 medium (Biological Industries, Beit Haemek, Israel) containing 10% fetal bovine serum (FBS, Biological Industries, Beit Haemek, Israel), A549 cells were cultured in F-12 K medium (Hyclone, Thermo Co., USA) supplemented with 10% FBS. All cells were cultured in a humidified incubator at 37 ℃ with 5% CO₂.
For vector and small interference RNAs (siRNA) transfection, jetPRIME reagent (polyplus-transfection SA) was used according to the manufacturer’s instructions. RPL11 overexpression vector were constructed by inserting the flag-conjugated RPL11 sequence into the GV141 vector. siRNA fragments of RPL11 (siRPL11) were synthesized by GenePharma (Shanghai, China). The siRPL11 sequences are si-1: Sense, 5’-GUAUGAUGGGAUCAUCCUU-3’, Antisense, 5’-AAGGAUGAUCCCAUCAUAC-3’; si-2: Sense, 5’-GCTUAGAUACACUGUCAGAU-3’, Antisense, 5’-AUCUGACAGUGUAUCUAGC-3’; si-3: Sense, 5’-GGUGCGGGAGUAUGAGUUA-3’, Antisense, 5’-UAACUCAUACUCCCGCACC-3’. The negative control siRNA (si-nc) sequences are Sense, 5’-UUCUCCGAACGUGUCACGU-3’, Antisense, 5’-ACGUGACACGUUCGGAGAA-3’.

All cell lines (A549, NCI-H1299, HCC827 and 16HBE) were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and confirmed by short tandem repeat (STR) profiling. HCC827 is a LUAD cell line with an acquired mutation in the EGFR tyrosine kinase domain (E746-A750 deletion), and 16HBE is a human bronchial epithelial cell line. The A549, NCI-H1299 and HCC827 cells were cultured in RPMI-1640 medium (Gibco, Invitrogen, Carlsbad, CA), and the 16HBE cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cell lines were grown in humidified air at 37 °C with 5% CO₂. The siRNA (si-hsa_circ_0005962, 5′-GAGACAACUUGACAUCUCUTT-3′) targeting the back-splice junction of hsa_circ_0005962 was synthesized by GenePharma (Shanghai, China). The A549 and H1299 cells were transfected with the siRNA using the Lipofectamine^® 2000 Reagent (Invitrogen) according to the manufacturer’s instructions. After transfection, the cells were processed to assess the knockdown activity by qRT-PCR or used for other experiments.

A549 and NCI-H460 cells were obtained from KeyGEN Bio TECH Corp., Ltd (Jiangsu, China). NCI-H1299 cells were obtained from Procell Life Science and Technology Co., Ltd (Hubei, China). A549 cells were cultivated within DMEM and F12 medium (Gibco, US), while NCI-H460 and NCI-H1299 cells were cultured within the RPMI 1640 medium (Gibco, US). 10% fetal bovine serum (FBS, Gibco, USA) and 100 U/ml penicillin-streptomycin mixed antibiotics were added into each medium. Each cell line was kept under 37°C in a humidified 5% CO₂ atmosphere.