Research Paper Volume 12, Issue 21 pp 21674—21686

Circular RNA circSEMA5A promotes bladder cancer progression by upregulating ENO1 and SEMA5A expression

Figure 4. CircSEMA5A serves as a miRNA sponge for miR-330-5p to upregulates ENO1 expression. (A) Diagram showed the potential binding site of miR-330-5p in circSEMA5A sequence. (B) Co-localization of circSEMA5A and miR-330-5p by FISH assays. (C) Luciferase reporter assay verified the predicted binding site between miR-330-5p and circSEMA5A. (D) The level of miR-330-5p pulled-down by circSEMA5A probe was analyzed by qRT-PCR. (E) The relative expression of miR-330-5p was detected by qRT-PCR in BC tissues and paired normal bladder tissues. (F) The relative expression of miR-330-5p was detected by qRT-PCR in BC cell lines and a normal human uroepithelial cell line. (G) Diagram showed the potential binding site of miR-330-5p in ENO1 3’UTR. (H) Luciferase reporter assay verified the predicted binding site between miR-330-5p and ENO1 3’UTR. (I) T24 cells were transfected with circSEMA5A overexpression plasmids and miR-330-5p mimics and UM-UC-3 cells were transfected with circSEMA5A siRNAs and miR-330-5p inhibitors, and ENO1 expression was detected by western blot assay. (J and K) Activations of Akt and β-catenin signaling pathways were evaluated by western blot assay. Data are presented as mean ± SD. *P < 0.05.