Cancer Res Treat.  2016 Oct;48(4):1302-1312. 10.4143/crt.2015.381.

TRIM29 Overexpression Promotes Proliferation and Survival of Bladder Cancer Cells through NF-κB Signaling

• Tan ST
• Liu SY
• Wu B

• KMID: 2356232
• DOI: http://doi.org/10.4143/crt.2015.381

Abstract

PURPOSE
TRIM29 overexpression has been reported in several human malignancies and showed correlation with cancer cell malignancy. The aim of the current study is to examine its clinical significance and biological roles in human bladder cancer tissues and cell lines.
MATERIALS AND METHODS
A total of 102 cases of bladder cancer tissues were examined for TRIM29 expression by immunohistochemistry. siRNA and plasmid transfection were performed in 5637 and BIU-87 cell lines. Cell Counting Kit-8, flow cytometry, western blot, and real-time polymerase chain reaction were performed to examine its biological roles and mechanism in bladder cancer cells.
RESULTS
We found that TRIM29 overexpression showed correlation with invading depth (p=0.0087). Knockdown of TRIM29 expression in bladder cancer cell line 5637 inhibited cell growth rate and cell cycle transition while its overexpression in BIU-87 cells accelerated cell proliferation and cell cycle progression. TRIM29 overexpression also inhibited cell apoptosis induced by cisplatin. In addition, we demonstrated that TRIM29 depletion decreased while its overexpression led to upregulated expression of cyclin D1, cyclin E, and Bcl-2. We also showed that TRIM29 knockdown inhibited protein kinase C (PKC) and nuclear factor κB (NF-κB) signaling while its overexpression stimulated the PKC and NF-κB pathways. BAY 11-7082 (NF-κB inhibitor) partly attenuated the effect of TRIM29 on expression of cyclin and Bcl-2. Treatment with PKC inhibitor staurosporine resulted in ameliorated TRIM29 induced activation of NF-κB.
CONCLUSION
The current study demonstrated that TRIM29 upregulates cyclin and Bcl family proteins level to facilitate malignant cell growth and inhibit drug-induced apoptosis in bladder cancer, possibly through PKC-NF-κB signaling pathways.

Keyword

TRIM29; NF-κB; Urinary bladder neoplasms; PKC

MeSH Terms

Apoptosis
Bays
Blotting, Western
Cell Count
Cell Cycle
Cell Line
Cell Proliferation
Cisplatin
Cyclin D1
Cyclin E
Cyclins
Flow Cytometry
Humans
Immunohistochemistry
Plasmids
Protein Kinase C
Real-Time Polymerase Chain Reaction
RNA, Small Interfering
Staurosporine
Transfection
Urinary Bladder Neoplasms*
Urinary Bladder*
Cisplatin
Cyclin D1
Cyclin E
Cyclins
Protein Kinase C
RNA, Small Interfering
Staurosporine

Figure

• 

  Fig. 1. Expression pattern of TRIM29 protein in bladder cancer tissues. (A) Negative TRIM29 staining in the majority of normal bladder urothelia. (B) Negative TRIM29 staining in bladder cancer (T1, grade 1). (C) Membrane staining of TRIM29 in urothelial bladder cancer (T2, grade 1). (D) Cytoplasmic staining of TRIM29 in bladder cancer (T2, grade 2).

• 

  Fig. 2. Expression of TRIM29 protein in bladder cancer cell lines. (A) Protein expression of TRIM29 in bladder cancer cell lines. The 5637 cell line shows relatively high expression and BIU-87 and SV-HUC-1 cell lines show relatively low expression. (B, C) Western blot and real-time polymerase chain reaction analysis showed that siRNA treatment markedly decreases TRIM29 levels in 5637 cells and TRIM29 transfection significantly increased its expression in BIU-87 cells.

• 

  Fig. 3. TRIM29 knockdown inhibited and TRIM29 overexpression promoted cell proliferation and cell cycle progression in bladder cancer cell lines. (A) Cell Counting Kit-8 (CCK-8) assay showed that TRIM29 knockdown inhibited proliferation in the 5637 cell line. Overexpression of TRIM29 promoted proliferation in the BIU-87 cell line. (B) TRIM29 knockdown increased G1 phase percentage and decreased S phase percentage in 5637 cells. Overexpression of TRIM29 increased S phase percentage and decreased G1 phase cells percentage in BIU-87 cells. (C) TRIM29 knockdown inhibited while its overexpression upregulated cyclin D1 and cyclin E expression at protein and mRNA levels.

• 

  Fig. 4. TRIM29 inhibited cisplatin induced cancer cell apoptosis. (A) Annexin V/propidium iodide analysis showed that TRIM29 knockdown increased apoptosis rate and its overexpression inhibited apoptosis. (B) Depletion of TRIM29 downregulated and its overexpression upregulated Bcl-2 expression in bladder cancer cell lines.

• 

  Fig. 5. TRIM29 regulates nuclear factor κB (NF-κB) signaling activity. (A) Western blotting and luciferase reporter analysis showed that knockdown of TRIM29 decreased the level of p-IκB and NF-κB luciferase activity in the 5637 cell line. Overexpression of TRIM29 led to increased p-IκB and NF-κB luciferase activity in BIU-87 cells. (B) NF-κB inhibitor BAY 11-7082 blocked NF-κB activity and the roles of TRIM29 in cancer cell proliferation, cyclin D1 and Bcl-2 upregulation in BIU-87 cells.

• 

  Fig. 6. TRIM29 regulates protein kinase C (PKC) signaling activity. (A) Western blotting analysis showed that knockdown of TRIM29 decreased while overexpression of TRIM29 increased p-PKC expression in bladder cancer cells. (B) PKC inhibitor staurosporine blocked the role of TRIM29 in p-IκB activity and in BIU-87 cells.

Reference

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