Figure 3. NNT-AS1 serves as a competing endogenous RNA (ceRNA) for miR-496 in bladder cancer cells. (A) Relative NNT-AS1 expression in nuclear and cytoplasmic fractions of T24 and TCC-SUP cells. (B) Bioinformatics prediction via starBase 3.0 uncovered two possible binding sites for miR-496 in NNT-AS1. (C) RT-qPCR was conducted to analyze miR-496 expression in T24 and TCC-SUP cells after introduction of either the miR-496 mimics or miR-NC. *P < 0.05 vs. group miR-NC. (D) Either plasmid wt-NNT-AS1 or mut-NNT-AS1 was cotransfected into T24 and TCC-SUP cells with either the miR-496 mimics or miR-NC for the measurement of luciferase activity. *P < 0.05 vs. the miR-NC group. (E) A RIP assay was carried out to determine the interaction between miR-496 and NNT-AS1 in T24 and TCC-SUP cells. *P < 0.05 vs. group IgG. (F) MiR-496 expression in 47 pairs of bladder cancer tissues and ANTs was assessed via RT-qPCR. *P < 0.05 vs. group ANTs. (G) The correlation between miR-496 and NNT-AS1 expression levels in the 47 bladder cancer tissue specimens was examined by Spearman’s correlation analysis. r = -0.6328, P < 0.0001. (H) The expression of miR-496 in NNT-AS1–depleted T24 and TCC-SUP cells was quantified by RT-qPCR. *P < 0.05 vs. siNC.