Wnt5a In Invasion And Metastasis Of Bladder Transitional Cell Carcinoma

Background:Bladder cancer is kind of a cancer happening on the sticky film of bladder, which is the most common cancer of urinary system and one of all over top-ten common tumors. Bladder cancer has the highest prevalence among the cancers over the world. The number of bladder cancer is7.6/10for the registered area in2012and ranked9th of the national malignant tumor prevalence. This disease may happen in any age, even in children. The incidence increases with age, and the highest incidence is the period of50～70years old. The risk in male is four times as high as that in female. The most common bladder cancer is endothelial carcinoma.A variety of facters and multi stages are involved in the development of bladder cancer. The regular cause of death is the invasion and metastasis of the tumor. Therefore, to explore the mechanisms of the growth of the bladder cancer, including invasion and metastasis could make important impact on the medical theory and clinical research.Previous studies on bladder cancer showed that valid lifetime is5years, but if there is invasion and metastasis happened during the bladder cancer, the formation period would be shorten to3years. Recently many researchers focussd on epithelial and mesenchymal transition of the bladder cancer invasion and metastasis. Epithelial and mesenchymal transition(EMT) is a basic physiological and pathological phenomenon, involved in embryonic development, tissue remodeling and tumor metastasis. Acquirement or deletion of characteristics of epithelial cell phenotype is its main feature.. More and more studies found Wnt family and its signaling pathway have close relationship with EMT. The Wnt family is a kind of glycoprotein that is rich in serine and consists of19members. There are receptors for the regulation of specific transcription factor on the cell surface. These receptors are involved in cell production, function regulation, cell polarization, and cell migration. Wnt5a is a member of the Wnt family that has attracted much attention in recent years. The studies have shown that it is closely related with the occurrence of bladder tumor and tumor invasion and metastasis. Some researches suggest that Wnt5a is a member of the Wnt family without transformation ability and mainly involved in the non canonical Wnt signaling pathway. The canonical Wnt signaling pathway is to facilitate tumor cell proliferation and to inhibit apoptosis which includes many members of the Wnt family for the development of tumor. During the tumor development, Wnts members appear different functions and unique way of expression by regulating the Wnt signaling pathway and play an important role in proliferation, apoptosis, migration and differentiation of tumor cells. Wnt5a is becoming a research spotlight because it is involved in growth and development of many diseases through many different mechanisms. Previous studies suggest that Wnt5a can increase the transformation between interstitial and epithelial, which is considered to be important in the pathological process of many tumors because of the abnormal expression in a variety of mesenchymal tumors. However, researchers also found some contradictory result that the expression of Wnt5a in different kinds of tumor tissues have different functions including both cancer inhibition and induction. Wnt5a appeared cancer gene characteristics. For example, in prostate cancer, non small cell lung cancer, breast cancer, gastric cancer, pancreatic cancer cells and so on. Because Wnt5a can facilitate tumor growth and enhance tumor invasion and metastasis ability. Nonetheless, the Wnt5a in colorectal cancer, leukemia and neuroblastoma cells can inhibit the development and metastasis of cancer, some studies suggested that Wnt5a can be regarded as an independent factor of poor prognosis. At present there is few researches on the relationship between Wnt5a expression and tumor development in bladder transitional cell carcinoma. [Aim]1. To investigate the relationship between Wnt5a factor and bladder cancer invasion and metastasis;2. To discover an effective method for inhibition of bladder cancer.[Content]1. To detect Wnt5a expression and significance in bladder transitional cell carcinoma using Immunohistochemistry and real-time PCR.2. Establishment of Wnt5a gene silencing and high expression in bladder cancer cell lines to explore the effect on invasiveness of bladder cancer cells through western blot, scratch repair experiments and Transwell chamber assay.3. To elucide the potential mechanism of Wnt5a in bladder cancer metastasis by western blot and real-time PCR test by dectecting epithelial markers (E-cadherin, N-cadherin) and mesenchymal markers (Snail, Twist). [Results]1. Immunohistochemistry assays showed the expression of Wnt5a increased gradually in bladder urothelial carcinoma, while real-time PCR detection found the highest expression of Wnt5a mRNA was in bladder cancer tissues, and the lower expression was in bladder cancer tissue. In papilloma tissues, the lowest expression existed in cancer tissues.2. Cell line J82with knock-down Wnt5a gene, western blot study proved the expression of Wnt5a protein was significantly decreased, which means Wnt5a was successfully knockdown. Cell scratch repair experiments showed the silence of Wnt5a gene led to the reduced mobility of bladder cancer cell lines. The Transwell chamber assay also found the invasion ability of bladder cancer cell line invasion decreased. The MTT assays shown that the knock down of Wnt5a gene resulted greatly decreased growth rate in bladder cancer cell lines.3. In the over-expression of Wnt5a in bladder carcinoma cell line BIU-87, Western blot assays showed the expression of Wnt5a protein increased significantly, which suggested the establishment of Wnt5a overexpression stable cell line. Scratch repair experiments found that over expression of Wnt5a enhanced the mobility of bladder cancer cell line. The Transwell chamber experiments showed that over-expression of Wnt5a increase the invasion ability of bladder cancer cell lines. The MTT assays found the bladder cancer cell line growth rate was increased by the overexpression of Wnt5a gene.4. Western blot and real-time PCR displayed that the expression of E-cadherin protein, N-cadherin protein and mRNA increased after Wnt5a knock-down, while the expression of Snail protein, twist protein and mRNA decreased. Western blot and real-time PCR showed over expression of Wnt5a would lead to opposite results.[Conclusion]1. The expression level of Wnt5a is closely related to the occurrence and invasion of bladder urothelial carcinoma, which indicated that Wnt5a would be used as a biomarker for potential treatment of bladder urothelial carcinoma,.2. Establishment of Wnt5a knockdown and overexpression in bladder cancer cell lines sucessfully.Wnt5a promoted the bladder cell proliferation、invasion and metastasis strongly, which suggested Wnt5a may be a new target for the therapy of bladder metastasis.3. Wnt5a would proment the transformation of epithelial mesenchymal in bladder cancer through affecting the EMT marked molecules of E-cadherin, N-cadherin, Snail, and Twist, therefore lead to invasion and metastasis of cancer cells.