Mir 431 5p mimic

Lung cancer cell lines (A549, H1975, SPC-A-1, H125, and H1299) and normal human lung cells (MRC-5) were provided by the American Type Culture Collection (ATCC; Manassas, VA, U.S.A.). All cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco/Invitrogen Inc., Carlsbad, CA, U.S.A.), streptomycin (100 mg/ml), and penicillin (100 U/ml), and then they were maintained at 37°C in humidified atmosphere with 5% CO₂.
For down-regulation of FBXL19-AS1 in cells, short hairpin RNA (shRNA) specifically targeting FBXL19-AS1 were designed and synthesized by GenePharma (Shanghai, China). The full-length sequences of FBXL19-AS1 and RAF1 were respectively synthesized and subcloned into pcDNA3.1 (Invitrogen, Carlsbad, U.S.A.) plasmid to produce pcDNA3.1/FBXL19-AS1 and pcDNA3.1/RAF1. MiR-431-5p mimic, miR-431-5p inhibitor, and the corresponding negative controls (miR-NC) were purchased from GenePharma. All plasmids were transfected into A549 and H1299 cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.) in reference of manufacturer’s recommendations. The sequences of shRNA were:
sh-FBXL19-AS1-1: 5′-CCG GCC TCC CTA AGT GTT GGG ATT ACT CGA GTA ATC CCA ACA CTT AGG GAG GTT TTT TG-3′;
sh-FBXL19-AS1-2: 5′-CCG GGC ATT TAA TTT GGC ATA GCA ACT CGA GTT GCT ATG CCA AAT TAA ATG CTT TTT TG-3′.

For cell treatment, A875 and SK-MEL-1 cells were exposed to ISI (I3766, Sigma, St. Louis, MO, USA) with increasing concentrations (10-80 μg/mL) for 24 h. For cell transfection, 1 × 10⁴ cells were planted into the 96-well plates and added with vectors or oligonucleotides via Lipofectamine™ 3000 kit (L3000001, Invitrogen, Carlsbad, CA, USA). The pcD5-ciR vector (GENESEED, Guangzhou, China) was cloned with circ_0002860 sequence for overexpression of circ_0002860 (oe-circ_0002860). The miR-431-5p mimic, miR-NC mimic, miR-431-5p inhibitor (anti-miR-431-5p), anti-miR-NC, small interfering RNA of RAB9A (si-RAB9A) and si-NC were commercially obtained from GenePharma (Shanghai, China).

The mouse lung epithelial (MLE‐12) cell lines were purchased from the BeNa Culture Collection. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), in a humidified atmosphere of 95% air‐ 5% CO₂ at 37°C. MLE‐12 cells were cultured in 6 well culture plate, and then the cells were transfected with miR‐205‐5p mimic, miR‐205‐5p inhibitor, miR‐431‐5p mimic, miR‐431‐5p inhibitor, NC or inhibitor negative control (INC), with lipofectamine 2000 for 48 h. miR‐205‐5p inhibitor, miR‐431‐5p inhibitor, INC were synthesized by Genepharma. These miRNA mimic (or respective inhibitor) transfected MLE‐12 cells were simultaneously infected with influenza A virus (MLE‐12 Infection model). The cells cultured in complete medium were considered as normal control. The culture plate was cultured in a 37°C, 5% CO₂ incubator. The cells were harvested 48 h after transfection, and Western blot was carried out using the primary antibodies against NP (Abcam) and GAPDH (Cell Signalling) as an internal control.²³