Research Paper Volume 11, Issue 24 pp 12624—12640

Long noncoding RNA NNT-AS1 enhances the malignant phenotype of bladder cancer by acting as a competing endogenous RNA on microRNA-496 thereby increasing HMGB1 expression

Figure 4. HMGB1 mRNA is a direct target of miR-496 in bladder cancer cells. (A) CCK-8 assay of the proliferation of T24 and TCC-SUP cells transfected with either the miR-496 mimics or miR-NC. *P < 0.05 vs. miR-NC. (B) The proportion of apoptotic miR-496–overexpressing T24 and TCC-SUP cells was detected by flow-cytometric analysis. *P < 0.05 vs. group miR-NC. (C, D) The migratory and invasive abilities were examined in transwell migration and invasion assays involving T24 and TCC-SUP cells transfected with either the miR-496 mimics or miR-NC. *P < 0.05 vs. the miR-NC group. (E) The predicted wild-type and mutant miR-496–binding sequences in the 3′-UTR of HMGB1. (F) A reporter plasmid containing either a wild-type or mutant HMGB1 3′-UTR fragment was cotransfected in combination with either the miR-496 mimics or miR-NC into T24 and TCC-SUP cells, and luciferase activity was quantified. *P < 0.05 vs. the miR-NC group. (G, H) Detection of HMGB1 mRNA and protein expression levels in miR-496–overexpressing T24 and TCC-SUP cells by RT-qPCR and western blot analysis, respectively. *P < 0.05 vs. group miR-NC. (I) HMGB1 mRNA expression was analyzed by RT-qPCR in the 47 pairs of bladder cancer tissue specimens and ANTs. *P < 0.05 vs. group ANTs. (J) Assessment of the correlation between miR-496 expression and HMGB1 mRNA expression among the 47 bladder cancer tissue specimens was performed via Spearman’s correlation analysis. r = -0.5221, P = 0.0002.