Fig 2: SORCS1 and CUBN are the functional targets of circSPIDR/miR-431-5p signaling. (A) Western blot analysis of SORCS1 and CUBN protein levels in CADC cells transfected with miR-431-5p inhibitors or the circSPIDR expression vector. (B) AGO2-RIP was performed using an anti-AGO2 antibody in HeLa cells transfected with circSPIDR or the vector. Then, qRT-PCR was used to assess and the enrichment of SORCS1 and CUBN. (C) Western blot analysis of SORCS1 and CUBN protein expression in CADC cells co-transfected with circSPIDR and miR-431-5p mimics. (D) CCK-8 assay in SORCS1- or CUBN-knockdown HeLa cells. (E, F) CCK-8 assay (E) and apoptosis assay (F) in HeLa cells following miR-431-5p inhibition and SORCS1 or CUBN knockdown. (G, H) CCK-8 assay (G) and apoptosis assay (H) in HeLa cells following circSPIDR overexpression and SORCS1 or CUBN knockdown. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: SORCS1 and CUBN are the target genes of miR-431-5p and circSPIDR. (A) miR-431-5p-target gene regulatory network. (B) qRT-PCR analysis of candidate target gene expression in CADC cells transfected with miR-431-5p inhibitors. (C, D) qRT-PCR analysis of candidate target gene expression in CADC cells transfected with the circSPIDR expression vector (C) or siRNAs against circSPIDR (D). (E) Luciferase activity of the 3'UTRs of SORCS1 and the CUBN in 293T cells co-transfected with miR-431-5p mimics. NS, not significant; *P < 0.05; **P < 0.01.