Figure 4. CircSPIDR directly binds to miR-431-5p as a miRNA sponge. (A) qRT-PCR analysis of miR-431-5p expression in 20 normal cervical tissues and 20 CADC tissues. (B) qRT-PCR analysis of miR-431-5p expression in HeLa cells transfected with circSPIDR, the vector, si-circSPIDR#1, si-circSPIDR#2 or si-NC. (C) RNA fluorescence in situ hybridization assay for circSPIDR and miR-431-5p in HeLa cells. (D) Schematic drawing showing the putative binding sites of miR-431-5p on circSPIDR. A luciferase reporter assay was performed to detect circSPIDR luciferase reporter activity in cells co-transfected with miR-431-5p mimics or miR-NC. (E) AGO2-RIP was conducted using an anti-AGO2 antibody in HeLa cells transfected with circSPIDR or the vector. The enrichment of miR-431-5p was then assessed using qRT-PCR. (F–G) CCK-8 (F) and colony formation (G) assays of HeLa cells transfected with miR-431-5p inhibitors. (H) Apoptosis analysis of HeLa cells transfected with miR-431-5p inhibitors. NS, not significant; *P < 0.05; **P < 0.01.