The Role Of NEDD9 In Bladder Cancer Metastasis

Backgroud:Bladder cancer is one of the most common malignant tumor in the urogenital system, which is characterized by its high frequent tumor recurrence. Currently, radical surgery based comprehensive therapy has been the accepted and popularized around the world, however, no breakthrough improvements were achieved to benefit the patients survival. The pathogenesis of bladder cancer has not been fully elucidated. At present, metastasis and recurrence of bladder cancer is considered to be the leading cause of bladder cancer death, which involves multiple genes and multi-level disorders related to cell signaling network, but the underlying molecular mechanism of bladder cancer metastasis is not yet fully understood. Therefore, to explore the molecular mechanism of bladder cancer occurrence, development and metastasis, is of great importance for identifying markers for early diagnosis and new therapeutic targets of bladder cancer.NEDD9 (Neural precursor cell Expressed Developmentally Down-regulated 9) is a kind of cytoskeleton proteins. NEDD9 was found to be overexpressed in a variety of malignant tumor tissues, and was proved to play an important role in tumor invasion and metastasis. Thus, NEDD9 is known as a molecule that is associated with tumor invasion and metastasis. But the role of NEDD9 in bladder is seldom reported at home and abroad. This study focus on the correlation between NEDD9 and bladder cancer development.Objective:Detect the NEDD9 mRNA and protein expression level in the bladder cancer tissue as well as in the normal bladder tissues, and further analyze its relationship with bladder cancer differentiation status. Then, the expression of NEDD9 were manipulated in bladder cancer cell line to observe the influence of NEDD9 on bladder cancer cell growth, apoptosis, transfer, migration and invasion.Method:1. The expression and significance of NEDD9 in bladder cancer tissueand adjacent tissuesCollect 24 paired cases of bladder cancer tissues, use RT-PCR to examine the expression of NEDD9 mRNA; immunohistochemistry (IHC) and western blot to examine the NEDD9 protein expression in various tissues and analyze the relationship between NEDD9 expression and bladder cancer differentiation status.2. The influence of NEDD9 on bladder cancer cell growth, apoptosis, migration and invasionUsing RT-PCR and western blot to investigate the NEDD9 expression in the bladder cancer cell line EJ, T24,5637, RT-4. Utilize NEDD9 overexpression adenovirus and NEDD9 siRNA to up-or down-regulate the NEDD9 expression in bladder cancer cell lines. MTT method, flow cytometry technology, wound scratch assay, migration and invasion assay were applied to observe the significance of NEDD9 bladder cancer cell growth, apoptosis, migration and invasion.Results:1. The expression and significance of NEDD9 in bladder cancer tissueIHC results showed that high and low differentiation bladder cancer tissues were dyed darker compared with normal bladder tissues, even more darker for poorly differentiated bladder cancer tissues in 24 cases of bladder cancer tissue samples. RT-PCR results revealed that NEDD9 mRNA expression level increased significantly in poorly differentiated bladder cancer tissues compared with tissue adjacent to carcinoma, but no significant difference were observed between highly differentiation bladder cancer tissue and normal bladder tissue. Western Blot results showed that NEDD9 protein expression were higher in poorly differentiated bladder cancer tissue.2. The influence of NEDD9 on bladder cancer cell growth, apoptosis, migration and invasionNEDD9 could be detected in bladder cancer cell lines EJ,5637, T24, RT-4 by both RT-PCR and western blot. The background expression level of NEDD9 were lowest in EJ and highest in 5637 among the four cells.In MTT assay,5637 cells growth were significantly inhibited after transfected with NEDD9 siRNA for 84h. The apoptosis rate of 5637-NEDD9 siRNA is about 10% higher than that of 5637-NC group, indicating that knock down NEDD9 in 5637 could inhibits cell proliferation and induce apoptosis.Upregulation of NEDD9 expression could promote the healing of EJ cells in wound scratch assay, can increase the number of cells in migration and invasion experiments, whereas 5637-NEDD9 siRNA cells showed reduced ability to heal scratches and a significant reduction in the number of cells which transfer through the basement membrane.Conclusion:1. the expression of NEDD9 in bladder cancer tissues is higher at both protein and mRNA level, and its expression level is closely related to bladder cancer differentiation status. These results indicate that NEDD9 might become a predictor of bladder cancer invasion and metastasis, and could be developed as an effective target for bladder cancer therapy.2. NEDD9 siRNA could significantly decrease NEDD9 expression in 5637 cells, thus inhibit cell proliferation, and induced apoptosis as well as block cell migration and invasion. Overexpression of NEDD9 by adenovirus can significantly enhace the level of NEDD9 expression in EJ cells, as a result, promote EJ cell migration and invasion.