Silencing of CARMA3 inhibits bladder cancer cell migration and invasion via deactivating β-catenin signaling pathway

Abstract

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.

Keywords: CARMA3; bladder cancer; invasion; migration; β-catenin signaling pathway.

Figure 1

CARMA3 was silenced efficiently in 5637 and T24 cells. (A) The levels of CARMA3 mRNA in 5637 and T24 cell were detected by real-time PCR (****p<0.0001). (B) The levels of CARMA3 expression in 5634 and T24 cells were assessed by Western blotting (****p<0.0001).

Figure 2

Knockdown of CARMA3 decreases the cell migration, invasion and the expression of MMP2 and MMP9 in 5637 and T24 cells. (A) Cells migration was performed by wound-healing assay in 5637 and T24 cells. bar =200 μm. (B) Cells invasion was assessed by transwell assay in 5637 and T24 cells, bar =100 μm. (C)The expression levels of MMP9 and MMP2 were evaluated by Western blotting in 5637 and T24 cells (**p<0.01, ***p<0.001). (D) MMP9 and MMP2 activities were analyzed by Gelatin zymography in 5637 and T24 cells (**p<0.01, ***p<0.001).

Figure 3

Silencing of CARMA3 deactivates β-catenin signaling pathway. (A) The distribution of β-catenin in 5637 and T24 cells was observed by immunofluorescence staining, bar =50 μm. (B) The mRNA level of β-catenin in 5637 and T24 cells was detected by real-time PCR (*p<0.05, **p<0.01). (C) The activity of β-catenin was examined by Luciferase assay (**p<0.01, ***p<0.001). (D, E and F) The expression of β-catenin, surviving and C-myc in 5637 and T24 cells was assessed by Western blotting (**p<0.01, ***p<0.001).

Figure 4

Silencing of CARMA3 inhibited the activity of adherens junction between the β-catenin and E-cadherin. (A) After transfection of si-NC, si-CARMA3 that targeted the 3ʹ-untranslated non-coding region of the CARMA3 mRNA, pcDNA3 or pcDNA3-CARMA3 into BC cells, the expression of CARMA3 and β-catenin was detected by Western blotting. (B) Cells migration was examined by wound-healing assay in 5637 and T24 cell after transfecting with empty vector or β-catenin S33Y plasmid, bar =200 μm. (C) Cells invasion was evaluated by Transwell in 5637 and T24 cell after transfecting with empty vector or β-catenin S33Y plasmid, bar =100 μm. (D) The expression of E-cadherin was examined by IF, bar =50 μm. (E) The interaction between β-catenin and E-cadherin was judged by Co-IP assay. ***p<0.001, ****p<0.0001.

Figure 5

Overexpressed β-catenin can reactivate β-catenin signaling pathway in silencing of CARMA3. (A) Cells migration was examined by wound-healing assay in 5637-sh-CARMA3 and T24-sh-CARMA3 cell before and after transfecting β-catenin S33Y plasmid, bar =200 μm. (B) Cells invasion was evaluated by Transwell in 5637-sh-CARMA3 and T24-sh-CARMA3 cell before and after transfecting β-catenin S33Y plasmid, bar =100 μm. (C) The expression levels of β-catenin, MMP9, MMP2 and C-myc were assessed by Western blotting in 5637 and T24 cells before and after transfecting β-catenin S33Y plasmid (**p<0.01, ***p<0.001).

Figure 6

Silencing of CARMA3 significantly affects tumor progression in vivo and decreases the expression of β-catenin. (A) T24 and 5637 cells transfected with sh-CARMA3 were injected subcutaneously in to nude mice (n=6) and the tumor growth was monitored every three days. (B) Comparison of immunohistochemical images of CARMA3 and β-catenin in the specimens corresponding to (A). (C) Western blotting examined the expression of CARMA3 and β-catenin in the specimens corresponding to (A) (****p<0.0001).