Cell proliferation assay

Cells seeded onto 24-well plates were first transfected with miR-138-5p or miR-204-5p mimics, inhibitors, the EGFR-overexpressing lentivirus, EGFR siRNA and the relevant negative control. At 24 h after transfection, EdU was added to the culture medium at a concentration of 50 µM/ml for 5 h to chase the DNA template. Briefly, after fixation in 4% paraformaldehyde and treatment with 0.5% Triton X-100 for 15 min, the cells were incubated in darkness with Apollo^®, and the nuclei were stained with DAPI using the Cell-Light EdU DNA cell kit (Apollo^® 567/488; Guangzhou RiboBio Co., Ltd., Guangzhou, China) according to the manufacturer's instructions. EdU-labeled and DAPI-labeled cells were manually counted in five fields randomly selected from each well, and the percentages were calculated. All experiments were performed in triplicate to do a statistical analysis.