Figure 3. VPS9D1-AS1 functions as a sponge for miR-532-3p in NSCLC cells. (A) Relative expression of VPS9D1-AS1 in nuclear and cytoplasmic fractions of H460 and A549 cells determined by subcellular fractionation and RT-qPCR analysis. (B) Predicted wild-type and mutant complementary sites of miR-532-3p in a VPS9D1-AS1 fragment. (C) RT-qPCR analysis of miR-532-3p expression in 51 pairs of NSCLC samples and corresponding normal lung tissues. *P < 0.05 vs. normal lung tissues. (D) Negative correlation between VPS9D1-AS1 and miR-532-3p levels in the 51 NSCLC tissue samples revealed by the Spearman’s rank correlation analysis. R2 = 0.3370, P < 0.0001. (E) RT-qPCR analysis of miR-532-3p expression levels in H460 and A549 cells with VPS9D1-AS1 knockdown. *P < 0.05 vs. the si-NC group. (F) RT-qPCR analysis of miR-532-3p expression levels in H460 and A549 cells transfected with agomir-532-3p or agomir-NC. *P < 0.05 vs. the agomir-NC group. (G) Interaction between miR-532-3p and VPS9D1-AS1 in NSCLC cells revealed by the luciferase reporter assay. H460 and A549 cells were co-transfected with a luciferase reporter plasmid carrying wild-type (wt) or mutant (mt) VPS9D1-AS1 and agomir-532-3p or agomir-NC. *P < 0.05 vs. the agomir-NC group. (H) miR-532-3p and VPS9D1-AS1 enrichment in AGO2 immunoprecipitates from H460 and A549 cell lysates determined by the RIP assay. IgG served as negative control. *P < 0.05 vs. the IgG group.