Fig 2: The overexpression of BRCC3 increased tumor cell proliferation in vitro. (A) BRCC3-overexpressing cells were identified via immunoblotting. (B) Colony formation assays. The number of clones was counted and plotted. (C) An ECIS cell proliferation assay was performed. (D) Three-dimensional colony formation assay was performed in soft agar. The colony numbers are expressed as the means ± standard error of triplicate assays. (E) The Quantitative data show the colonies numbers in (D). (F) Transwell migration assay. The relative cell numbers of migrating were statistically analyzed. (G) Quantitative data show the number of migrating cells in (F). *P< 0.05, **P< 0.001, and ***P< 0.001 compared with controls.

Fig 3: Knocking out of BRCC3 resulted in inactivation of the NF-κB signaling pathway. (A) KEGG pathway enrichment analysis was used to assess wild type and BRCC3–/– bladder cancer cells. (B) GSEA identified NF-κB pathway-related gene sets enriched in the wild-type cells. (C) BRCC3 promotes the transcriptional activity of NF-κB. 293T cells were transfected with an NF-κB reporter firefly luciferase plasmid (200 ng), pRL-TK (10 ng) and the indicated amounts of a BRCC3 plasmid. Reporter assays were performed 48 h after transfection, and the results are presented as the NF-κB/TK luciferase activity. Data were analyzed employing one-way ANOVA and presented as the means ± standard error (n = 3/group). (D) BRCC3 promoted the transcriptional activity of NF-κB in bladder cancer 5637 bladder cancer cells. The experiments and data analyses were performed as in (C). (E) BRCC3 ablation inhibited the transcriptional activity of NF-κB. (F) The BRCC3 deficiency blocked the transcription of NF-κB-targeted genes in the T24 cells. The expression levels of the indicated NF-κB-targeted genes were examined by RT-qPCR in BRCC3–/– T24 cells and wild type T24 cells. (G) BRCC3 deficiency reduced the degradation of IκBα under TNFα stimulation. BRCC3–/– T24 cells and wild type T24 cells were treated with 5 ng/ml TNFα for indicated time, and then the cell total protein lysis was analyzed by immunoblotting assays. (H) Quantitative data show the relative protein levels of IκBα in (G). Statistical analysis was conducted using a t-test. The means ± standard error from three independent experiments is shown. *P< 0.05, **P< 0.001, and ***P< 0.001 compared with controls.

Fig 4: The depletion of BRCC3 inhibits bladder cancer tumorigenesis in vivo. (A) Xenograft models (n = 6) were established by subcutaneously inoculating wild-type or BRCC3-KO cells and allowing the cells to grow for 5 weeks. Then the mice were sacrificed and the tumors were removed and weighed. (B–D) The tumor volume and weight measurements are shown. (E) Representative H&E staining and immunohistochemical staining of the xenograft tumors from the tumor-bearing mice in the wild-type and BRCC3-KO groups, showed the deficiency of BRCC3 (the fifth panel) and the upregulation of IκBα expression (the second panel), the downregulation of Ki67 expression (the top panel), TNFα expression (the third panel) and p-P65 expression (the fourth panel) in the BRCC3-KO groups. Statistical analysis of the tumor volume and weight were performed using one-way ANOVA. The means ± standard error of three independent experiments is shown. *P < 0.05, **P < 0.001, and ***P < 0.001 compared with controls.

Fig 5: Our study demonstrated that BRCC3 could promote BCa cells proliferation and migration both in vitro and in vivo. Furthermore, BRCC3 exerts its oncogenic role via binding to TRAF2, which in turn activates NF-κB signaling and inflammation in bladder cancer.