Research Paper Volume 12, Issue 21 pp 21971—21991

Figure 5. STAP1 is directly targeted by miR-587 and serves as a regulator in IDD. (A) Heat map of all differentially expressed mRNAs between the sample with CircGLCE knockdown and the paired controls. (B) The RT-qPCR results showed that STAP1 was downregulated in degenerative NP tissue samples (degenerative vs. normal=16: 22, ***P < 0.001, unpaired two-tailed Student’s t test). (C) Venn diagram displaying miR-587 computationally predicted to target SIRT1 by different algorithms. (D) Schematic representation of STAP1 3’-UTR demonstrating putative miRNA target site, luciferase activities of wild-type (WT-UTR), and mutant (MUT-UTR) constructs. (E) After co-transfection with the miR-587 mimics or control and the luciferase reporter constructs for wild-type or mutant 3′-UTR of STAP1, the binding of STAP1 and miR-587 was detected by dual luciferase assays (n=6, *** P<0.001, unpaired two-tailed Student’s t test). (F) The altered expression patterns of STAP1 protein were caused by miR-587 overexpression or silencing, which was detected by western blotting (n=3). (G) The altered expression patterns of STAP1 mRNA were caused by miR-587 overexpression or silencing, which was detected by RT-qPCR (n=6, **P < 0.01, and ***P < 0.001; unpaired two-tailed Student’s t test). (H) The FISH results of the rescue experiment using the pre-miR-587 adenovirus, STAP1 and the control. (I) The results of RT-qPCR (n=3, ***P < 0.001, one-way ANOVA coupled with Tukey’s post hoc test) confirmed the FISH results in (H). (J) Western blot analysis (n=3) confirmed the results in (H and I). Data are the mean ± SEM.