Journal: Journal of Translational Medicine
Article Title: The biomarker potential of circPOLD1 and its binding protein YBX1 in cervical carcinogenesis
doi: 10.1186/s12967-025-06494-3
Figure Lengend Snippet: CircPOLD1 promotes the proliferation through directly binding to YBX1 in cervical cancer cells. A Schematic representation of the sites of the siRNAs specific to the back-splice junction of circPOLD1. B The circPOLD1 and POLD1 mRNA expression measured by qRT-PCR analysis in SiHa and CaSki cells after transfected with two siRNAs. Data were the means ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test). C The proliferation measured by CCK-8 assay in SiHa and CaSki cells transfected with circPOLD1 siRNAs, oligo control (oligo) and without treated (wildtype). Data were the means ± SEM of three experiments (*P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test). D Representative image (left) and quantification (right panel) of colony formation assays in SiHa and CaSki cells transfected with circPOLD1 siRNAs or oligo control. Data were the means ± SEM of three experiments (*P < 0.05, **P < 0.01, Student’s t-test). E Apoptotic rates measured by flow cytometry in SiHa and CaSki cells transfected with circPOLD1 siRNAs or oligo control. Data were the means ± SEM of five experiments (*P < 0.05, **P < 0.01, Student’s t-test). F The circPOLD1 expression measured by qRT-PCR in SiHa cells after stably transfected with circPOLD1 shRNA(sh-circPOLD1) or oligo shRNA(oligo). G SiHa cells with stably transfected sh-circPOLD1 or oligo were subcutaneously injected into 6 BALB/c nude mice (4–6 weeks old, female), respectively, to establish the xenograft models. Top panel: The growths of xenografts were compared between sh-circPOLD1 and oligo group (*P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test). Tumor volumes were measured per week. Bottom panel: The photo showed the sizes of tumors when mice were sacrificed at week 8. H YBX1, the circPOLD1 binding protein predicted by online web server RBPmap. I , J Probes targeting back-splice junction of circPOLD1 and control oligo were transcribed in vitro, biotinylated, captured by streptavidin magnetic beads, and incubated with CaSki whole-cell lysates for RNA pull-down assays. The expression level of RNA measured by qRT-PCR in the eluate (left). CircPLD1 specific bands were assessed by Western blotting (right). Data were the means ± SEM of three experiments (*P < 0.05, Student’s t-test). K RIP assays were performed using antibodies against YBX1 in CaSki cells. The quantity of circPOLD1 was evaluated by qRT-PCR. Data were the means ± SEM of three experiments (*P < 0.05, Student’s t-test). L The expression level of YBX1 protein in SiHa cells after treated with two YBX1 siRNAs. M The proliferation by CCK-8 assay in SiHa and CaSki cells transfected with oligo control or YBX1 siRNAs. Data were the means ± SEM of three experiments (*P < 0.05, **P < 0.01, One-way ANOVA). N Representative images (left) and quantification (right) of colony formation assays in SiHa and CaSki cells transfected with YBX1 siRNAs or control. Data were the means ± SEM of three experiments (*P < 0.05, **P < 0.01, One-way ANOVA). O Co-transfection with circPOLD1 siRNA (si-circPOLD1) and YBX1 plasmid in CaSki cells was established. Down-regulated circPOLD1 expression and up-regulated YBX1 expression were evaluated by qRT-PCR. P Relative mRNA expressions of three downstream target genes of YBX1 (CDC25A, CIAPIN1 and XIAP) were measured by qRT-PCR in CaSki cells treated with control vector, si-circPOLD1, and si-circPOLD1 plus YBX1, respectively. Q The proliferation measured by CCK8 assay in CaSki cells treated with control vector, si-circPOLD1, and si-circPOLD1 plus YBX1, respectively. R Representative images of colony formation assays in CaSki cells treated with control vector, si-circPOLD1 and si-circPOLD1 plus YBX1, respectively. Data were the means ± SEM of three experiments (*P < 0.05, **P < 0.01, One-way ANOVA)
Article Snippet: Human cervical cancer SiHa cell line was obtained from the American Type Culture Collection (ATCC, USA).
Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Transfection, CCK-8 Assay, Control, Flow Cytometry, Stable Transfection, shRNA, Injection, In Vitro, Magnetic Beads, Incubation, Western Blot, Cotransfection, Plasmid Preparation
