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Xiaojun Man,1,2 Tao Liu,1,2 Yuanjun Jiang,1,2 Zhe Zhang,1,2 Yuyan Zhu,1,2 Zhenhua Li,1,2 Chuize Kong,1,2 Jiani He31Department of Urology, The First Hospital of China Medical University, Shenyang 110001, People’s Republic of China; 2Institute of Urology, Department of Urology, China Medical University, Shenyang 110001, People’s Republic of China; 3Breast Division, Department of Surgical Oncology, The First Hospital of China Medical University, Shenyang 110001, People’s Republic of ChinaBackground: Bladder cancer (BC) is the ninth most common cancer and the fourteenth leading death worldwide. CARD-containing MAGUK 3 (CARMA3) protein is a novel scaffold protein known to activate NF-κB pathway and is overexpressed in BC tissues.Purpose: The objective of this study was to identify how CARMA3 affects the metastasis of BC cells via the β-catenin signaling pathway.Materialsand methods: In the present study, 5637 and T24 BC cells with stable low expression of CARMA3 were established, and their migratory and invasive capabilities were further evaluated by wound-healing and transwell assay. The activity and expression of β-catenin were determined by Luciferase assay and immunofluoresence staining. The mRNA and protein expression levels of CARMA3, matrix metallopeptidase (MMP) 9 and MMP2 were detected by quantitative real-time PCR (qRT-PCR) and Western blot analysis. The nude mouse tumor xenograft model was established for in vivo study.Results: By comparison to the control cells, CARMA3-silenced cells acquired a less aggressive phenotype: decreased migration and invasion. More importantly, we confirmed that CARM3 knockdown could inhibit β-catenin mRNA and protein expression and activity, and reduce the expression and/or activity of matrix metallopeptidase (MMP) 9, MMP2 and C-myc. Also, CARM3 silencing increased E-cadherin expression and attenuated the expression of β-catenin. Moreover, we demonstrated that β-catenin overexpression reversed the inhibiting effect of CARMA3 silencing on cell invasion and migration. Furthermore, our study illustrated that knockdown of CARMA3 suppressed BC cells xenograft tumor growth in nude mice.Conclusion: We demonstrated that CARMA3 contributes to the malignant phenotype of BC cells at least by activating β-catenin signaling pathway, and it may serve as a therapeutic target for clinic treatment in BC.Keywords: CARMA3, β-catenin signaling pathway, bladder cancer, migration, invasion &nbsp

Figure S4. The image of silver staining to visualize the binding between USP13 and PTEN protein, and the enrichment of PTEN protein after NF-kB activation. 293 T cells were transfected with Flag-tagged USP13 (lane 2 and 3) or empty vector (lane 1) for 24 h, then followed by TNF-a (lane 2) or relative vehicle treatment (lane 1 and 3) for 12 h. Lysates were subjected to immunoprecipitation with anti-Flag M2 beads. The pull-down production was then subjected to silver staining assay. In lane 3, band could be visualized at around 50 kD (PTEN), and the band at 50 kD was significant strengthened after NF-kB activation (lane 2). Bands at around 95 kD (USP13) could also be visualized in lane 2 and 3. (PDF 2701 kb)

Figure S2. Cell proliferation index was measured in BC cells after knockdown of miR-130b/301b by CCK-8 assay (a and b) and colony formation assay (c and d). Cell invasive and migrative capacities were measured in BC cells after knockdown of miR-130b/301b by transwell assay (e and f). Number of colonies and invaded/migrated cells was counted by Image J software. Original magnification: 400��. *P

Figure S1. a. The schematic diagram of mir-130b and mir-301b gene. b. Expression of USP13 in normal bladder tissues and bladder tumor tissues from TCGA dataset. c. Expression of USP13 in normal bladder tissue and bladder tumor tissues based on individual stages (TCGA). d and e. Expression of hsa-miR-130b-3p and hsa-miR-301b-3p in normal bladder tissue and bladder tumor tissue from TCGA dataset. (PDF 724 kb)

Figure S3. USP13 expression was measured by western blot analysis (a) and qRT-PCR analysis (b) after USP13-shRNA transduction in 5637 cells. c. The schematic diagram of NF-kB binding site on the promoter region of mir-130b~301b cluster. d and e. Expression of NF-kB downstream targets was detected 0, 6, 12, 24 h after TNF-�� treatment in 5637 and UM-UC-3 cells. f and g. expression of miR-130b-3p and miR-301b-3p was detected 0, 6, 12, 24 h after TNF-�� treatment in 5637 and UM-UC-3 cells. h. NF-kB p65 was overexpressed in 5637 and UM-UC-3 cells by transfecting the pLvx-NF-kB p65 plasmids into the cells. i and j. Expression of NF-kB target genes, miR-130b-3p and miR-301b-3p was detected in BC cells transfected with pLVX-NC or pLVX-NF-kB p65. k. Western blotting analysis was performed to measure the expression of USP13 in response to Flag-USP13 plasmids transfection in 5637 cells. For real-time PCR, ��-actin and U6 snRNA were used as the internal control for mRNA and miRNA, the Ct values for each group were compared using the the 2-����Ct method. The internal control genes were GAPDH for western blot analysis, and the gels were run under the same experimental conditions. The band intensities were calculated by Image J 1.46r software, and the ratio of target gene to GAPDH was used to conduct the statistical analysis. *P

Background Bladder cancer (BC) is the ninth most common cancer and the fourteenth leading death worldwide. CARD-containing MAGUK 3 (CARMA3) protein is a novel scaffold protein known to activate NF-κB pathway and is overexpressed in BC tissues. Purpose The objective of this study was to identify how CARMA3 affects the metastasis of BC cells via the β-catenin signaling pathway. Materials and methods In the present study, 5637 and T24 BC cells with stable low expression of CARMA3 were established, and their migratory and invasive capabilities were further evaluated by wound-healing and transwell assay. The activity and expression of β-catenin were determined by Luciferase assay and immunofluoresence staining. The mRNA and protein expression levels of CARMA3, matrix metallopeptidase (MMP) 9 and MMP2 were detected by quantitative real-time PCR (qRT-PCR) and Western blot analysis. The nude mouse tumor xenograft model was established for in vivo study. Results By comparison to the control cells, CARMA3-silenced cells acquired a less aggressive phenotype: decreased migration and invasion. More importantly, we confirmed that CARM3 knockdown could inhibit β-catenin mRNA and protein expression and activity, and reduce the expression and/or activity of matrix metallopeptidase (MMP) 9, MMP2 and C-myc. Also, CARM3 silencing increased E-cadherin expression and attenuated the expression of β-catenin. Moreover, we demonstrated that β-catenin overexpression reversed the inhibiting effect of CARMA3 silencing on cell invasion and migration. Furthermore, our study illustrated that knockdown of CARMA3 suppressed BC cells xenograft tumor growth in nude mice. Conclusion We demonstrated that CARMA3 contributes to the malignant phenotype of BC cells at least by activating β-catenin signaling pathway, and it may serve as a therapeutic target for clinic treatment in BC.

Background: It is now widely recognized that there is a strong correlation between oxidative stress and the risk of benign and malignant diseases of the prostate. Prostate-associated gene 4 (PAGE4) is a Cancer/Testis Antigen (CTA) that was previously shown to be up-regulated in prostate cancer (PCa) and symptomatic as opposed to histologic benign prostatic hyperplasia (BPH). However, its functional role in these diseases is not fully understood. Methods: The mRNA level of PAGE4 was detected in isolated cell types in PCa tissues that were obtained from 8 men with PCa. PAGE4 protein expression profile was analyzed in a prostate disease tissue microarray. PAGE4 was overexpressed by pCMV-PAGE4-GFP transfection and cell viability was determined using the WST-1 assay. Results: PAGE4 expression is highly dynamic; while its expression is very high in fetal prostate it is drastically decreased in the normal adult prostate but is up-regulated both in symptomatic BPH and PCa. However, in the diseased prostate, PAGE4 is highly expressed in the epithelial cells of Proliferative Inflammatory Atrophy (PIA) lesions alluding to a potential stress response function of PAGE4. Consistent with such a role, PAGE4 protein levels are up-regulated when prostate cancer (PCa) cell lines are treated with various stress factors including the proinflammatory cytokine TNFα. Interestingly, in cells challenged with stress there is increased translocation of the PAGE4 protein to the mitochondrion and production of reactive oxygen species is suppressed . Furthermore, p21 is elevated in a p53-independent manner in PAGE4-overexpressing cells which results in impeded cell cycle progression, attenuated stress-induced DNA damage, and decreased cell death. Conclusions: PAGE4 may be contributing to the development of PCa by playing a stress-protective and anti-apoptotic role.