Fig. 3

circPVT1 promotes the migration and invasion of NPC cells by binding to β-TrCP. A. LC–MS/MS were performed to identify circPVT1 interacting proteins in CNE2 cells after pull-down with biotin-labeled circPVT1 probe. The unlabeled circPVT1 probe was used as control. B. Binding of circPVT1 and β-TrCP protein was analyzed in CNE2 and 5-8F cells after RNA pull-down with biotin-labeled circPVT1 probe. The biotin-labeled scrambled sequences was used as a control. C. Direct binding of β-TrCP protein to circPVT1 was evaluated in CNE2 and 5-8F cells by RNA immunoprecipitation using anti-β-TrCP antibody, followed by qRT-PCR analysis of circPVT1. Data were represented as mean ± SD. ***, p < 0.001. D. The 230–280 nt of circPVT1 was crucial for the interaction between circPVT1 and β-TrCP proteins. CNE2 and 5-8F cells were transfected with the full-length circPVT1 (circPVT1) or the 230–280 nt deleted mutant (△circPVT1). RNA pull-down assays were performed using biotin-labeled circPVT1 probe, followed by western blotting using anti-β-TrCP antibody. E. β-TrCP protein directly binds to the 230–280 nt of circPVT1 in CNE2 and 5-8F cells. Cells were were transfected with the full-length circPVT1 or the deletion mutant (△circPVT1). RNA immunoprecipitation was performed using anti-β-TrCP antibody, followed by qRT-PCR of circPVT1. Data were represented as mean ± SD. ***, p < 0.001, ns, not significant. F. The binding between circPVT1 and the WD40 domain of β-TrCP protein was examined in CNE2 and 5-8F cells after transfected with Flag-tagged full-length β-TrCP or truncated mutants (F-box or WD40). RNA pull-down assays were performed using biotin-labeled circPVT1 probe, followed by western blotting using anti-Flag antibody. G. The binding between circPVT1 and the WD40 domain of β-TrCP protein was examined in CNE2 and 5-8F cells. After transfected withFlag-tagged full-length β-TrCP or truncated mutants (F-box or WD40), RNA was immunoprecipitated using anti-Flag antibody. IgG was used as a control. Data were represented as mean ± SD. ***, p < 0.001, ns, not significant. H. Wound healing assay showed that overexpression of β-TrCP reverses the migrative ability of cicPVT1 in NPC cells. Data were represented as mean ± SD. ***, p < 0.001, ns, not significant. I. Transwell assay showed that overexpression of β-TrCP reverses the invasive ability of cicPVT1 in NPC cells. Data were represented as mean ± SD. ***, p < 0.001, ns, not significant