Cell culture and transfection
Human non-small cell lung cancer cell lines (A549 and H1299) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Hyclone, USA), 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37〬C with 5% CO2 atmosphere. Lipofectamine2000 liposome was used to transfect siRNA or plasmid into cells following the instructions. siRNAs targeting to OGFRP1 (siOGFRP1) were designed and synthetized (RiboBio, Guangzhou, China). miR-4640-5p mimic was purchased from RiboBio (Guangzhou, China). The cDNA of eIF5A1 was synthesized by GENEWIZ and cloned into the pcDNA3.1 expression vector (GenePharma, Shanghai, China).
Total RNA was extracted by using TRIzol (Invitrogen) according to the manufacturers’ instructions. The cDNA was formed by using EasyScriptTM Reverse Transcriptase (TransGen Biotech Co., Ltd., Beijing, China). The mRNA expression was further detected by using an FTC-300 Real-Time Quantitative Thermal Cycler (Funglyn Biotech Inc., Shanghai, China). GAPDH was used as an internal reference.
After 24 h of transfection, the cells were digested, resuspended and counted. 1000 cells were planted in each well of a 96-well plate. Cell viability was measured every 24 hours. For testing, 10 µl of CCK8 reagent was added to each well of the 96-well plate, and incubated at 37 °C for 2 h. Then the OD value at 450 nm was measured to draw the proliferation curve.
Wound healing migration assay
After transfection for 24 h, cells were scratched by a sterile pipettes tip and washed by PBS to eliminate suspended cells. Subsequently cells were cultured in fresh medium and photographed at 0 h and 24 h.
The transwell chamber was coated with Matrigel. The cells that had been transfected for 24 hours were prepared into a cell suspension with serum-free medium. 100 µl of cell suspension containing 1 × 104 cells was added to the upper chamber, and 600 µl of medium containing 10% FBS was added to the lower chamber. After culturing for 24 hours, the residual cells on the upper chamber were removed and washed with PBS. Then the cells on the lower surface of the chamber were fixed with paraformaldehyde for 15 min and stained with 0.1% crystal violet for 5 min. After washed with PBS, the cells were photographed and counted under a microscope.
After 48 h of transfection, the total proteins of the cells were extracted using RIPA buffer. 20 µg of proteins were taken for SDS-PAGE electrophoresis, and then electrotransferred onto a PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 h, incubated with the specific primary antibody at 4 °C overnight and incubated with the second antibody for 1 h at room temperature. ECL development was performed after washing the membrane through TBST.
Luciferase reporter assay
The complete 3'UTR of human eIF5A1 mRNA containing the putative or mutated miR-4640-5p binding site, and the wild or mutated full-length sequence of OGFRP1 were amplified and cloned into the psiCHECK2 vector (Promega). According to the manufacturer's guidelines, Lipofectamine 2000 was used to co-transfect psiCHECK2 recombinant vector and miR-4640-5p mimic or miR-NC into A549 cells. The relative activity of luciferase was measured using the Dual-Luciferase Reporter Assay System (Promega) and the Infinate M200 PRO microplate reader (Tecan, Shanghai, China).
All data were statistically analyzed using SPSS software version 22.0 (IBM Corp., Armonk, NY). All results were expressed as mean ± standard deviation. The difference between groups was calculated using the Student's t-test or one-way ANOVA. P < 0.05 was considered statistically significant.