A549 cells

Human bronchial epithelial cells (HBE) were obtained from FUHENG Biology (Shanghai, China). PLA-801D, NCI-H1299, HCC827, NCI-H1437 and NCI-H446 cells (Procell, Wuhan, China) were maintained in RPMI-1640 (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA). A549 cells (Procell) were grown in Ham's F-12K medium (Procell) containing 10% FBS. HEK-293T cells (Zhongqiaoxinzhou, Shanghai, China) were maintained in DMEM (Gibco) containing 10% FBS. All cells were cultured in a humidified atmosphere with 5% CO₂ at 37°C.
For transfection, circ_0089823 siRNAs (si-circ_0089823) and their negative control (si-NC), circ_0089823 shRNA (sh-circ_0089823) and its negative control (sh-NC) were transfected into A549 cells, while circ_0089823 over-expression plasmid (OE-circ_0089823) and its negative control (vector) were transfected into PLA-801D cells using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the protocol. Similarly, microRNA mimics, inhibitors and their negative controls (mimics NC and inhibitor NC) were transfected into A549 cell and PLA-801D cells using Lipofectamine 2000 Reagent. Cells transfected with sh-circ_0089823, sh-NC, OE-circ_0089823 or vector were selected with G418 (350–400 μg/ml; Solarbio, Beijing, China) to obtain stably transfected cell clones.

Lung squamous cell carcinoma cell line (H2170 cells) (product number: CL-0394), lung adenocarcinoma cell line (A549 cells) (product number: CL-0016), and human embryonic lung cell line (MRC-5 cells) (product number: CL-0161) were obtained from Procell (Wuhan, China) and orderly maintained in Roswell Park MEMorial Institute (RPMI)-1640 medium (Gibco, Grand Island, NY, USA), Ham’s F-12K (Thermo Fisher Scientific, Waltham, MA, USA), or Minimum Essential Medium (MEM) (Thermo Fisher Scientific) plus 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C and 5% CO₂. The cell culture medium was replaced when cells were cultured for 3–4 days.

A549 cells (Procell, Wuhan, China) were cultured in RPMI-1640 (Gibco, Sigma Aldrich, Denmark) containing 10% fetal bovine serum (FBS, Gibco, Sigma Aldrich, Denmark), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Sigma Aldrich, Denmark) at 37 °C. Cells were digested with 0.25% trypsin (Gibco, Sigma Aldrich, Denmark) and passaged at 80% confluence.

BEAS-2B (Human bronchial epithelial cells), NCI-H1299 cells, and A549 cells (human NSCLC cell line) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China), and were cultured in a 37 ℃ and 5% CO₂ constant-temperature incubator with RPMI-1640 medium containing 10% fetal calf serum (HyClone, ThermoFisher, Shanghai). Every second day, the cells were digested with typsin and subcultured, and during their logarithmic growth period, were transfected according to the following protocol.
The miR-137 mimic, mimic negative control, miR-137 inhibitor, inhibitor negative control, COX-2 siRNA, and scramble siRNA were synthesized by RiboBio Co., Ltd (Guangzhou, China). The coding sequence of COX-2 was cloned into lentivirus vector GV492 to obtain the recombined overexpression vector Lv-COX-2, which was provided by Vigen Biotechnology (Zhengjiang, China). The mimic, inhibitor, or siRNA was transfected into H1299 or A549 cells with lipofectamine 2000 (Invitrogen, Shanghai, China). In brief, the cells were digested with typsin and cultured into a 6-well plate with 50,000 cells/well before 24 hours of transfection. The miR-137 mimic, inhibitor (50 pmol), or COX-2 siRNA (100 pmol) were diluted in OPTI-MEM and transfected into NSCLC cells, and Lv-COX-2 was transfected into A549 cells at a multiplicity of infection (MOI) of 5.