Fig. 6

circPTPN4 functions as a competing endogenous RNA (ceRNA) to regulate NAMPT expression by sponging miR-499-3p. (A) The potential binding site of miR-499-3p in circPTPN4 transcript and NAMPT 3′ untranslated region (UTR). The mutant sequence in miR-499-3p binding site is highlighted in red. (B and C) Dual-luciferase reporter assay was conducted by co-transfecting the wild type or mutant: (B) circPTPN4 and (C) NAMPT 3′ UTR with a miR-499-3p mimic or mimic-negative control (NC). (D) The interaction of miR-499-3p with circPTPN4 and NAMPT was determined by biotin-coupled miRNA pull down. (E) Relative miR-499-3p, circPTPN4, and NAMPT expression after overexpression of miR-499-3p. (F and G) Relative mRNA (F) and protein (G) expression levels of NAMPT with circPTPN4 overexpression. (H and I) Relative mRNA (H) and protein (I) expression levels of NAMPT after circPTPN4 interference. In panels (G and I), the numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to β-Tubulin. Data are expressed as a fold-change relative to the control. Results are presented as mean ± SEM. In panels (B to H), the statistical significance of differences between means was assessed using independent sample t-test. (*P < 0.05; **P < 0.01)