Figure 3. NR2F1-AS1 directly interacts with miR-493-5p in NSCLC cells as a ceRNA. (A) The subcellular distribution of NR2F1-AS1 in the cytoplasm or nucleus of H460 and A549 cells. (B) The wild-type binding site of miR-493-5p in NR2F1-AS1 was predicted by StarBase 3.0. The mutant binding sequences were also shown. (C) The efficiency of miR-493-5p mimic transfection in H460 and A549 cells was examined by RT–qPCR. MiR-NC served as the control. (D) The wt-NR2F1-AS1 or mut-NR2F1-AS1 reporter plasmids alongside miR-493-5p mimic or miR-NC were cotransfected into H460 and A549 cells. Luciferase reporter assay indicated that miR-493-5p could directly bind to NR2F1-AS1 in H460 and A549 cells. (E) RIP assay revealed the enrichment of NR2F1-AS1 and miR-493-5p in RNA immunoprecipitation with AGO2 antibody. (F) MiR-493-5p expression in 73 pairs of NSCLC tissues and matched adjacent normal tissues was determined via RT–qPCR. (G) Pearson's correlation coefficient was utilized to test the relationship between NR2F1-AS1 and miR-493-5p in the 73 NSCLC tissues (r = −0.6435, P < 0.0001). (H) MiR-493-5p expression was analyzed by RT–qPCR in H460 and A549 cells after transfection with si-NR2F1-AS1 or si-NC. *P < 0.05 and **P < 0.01.