Research On The Detection Of Robos Expression In Bladder Cancer Cells And The Influence When Its Be Silenced

BackgroundBladder cancer, pathogenic mechanism unclear recently, is a common malignancy ofurinary surgery. There is no specific and useful therapies except early diagnosis andsurgical removal. It was firstly found that Slit family, a sort of neural guidance factors,plays a key role in regulations of the directional migration of neuronal precursors and thedirectional growth of neuron axons. Recent studies show that Slit/Robo signal pathway isinvolved in the regulation of the migration of tumor cells and the production of tumorvessels. Additionally, Robo is found express in tumor cells of breast cancer and livercancer in a few other studies. All these studies prove that Slit/Robo signal may well bevital signal pathway of tumorigensis and tumor metastasis.In order to further investigate the function of Robo signal protein in pathologies, andfind out therapeutic methods of bladder cancer, Robo signal expression will be detected inhuman transitional epithelial cancer cell line of bladder T24, cultured in vitro. If Roboexpression is positive in T24cells, silence this signal pathway with RNA interferencetechnique and observe following growth condition.Methods1. Immunohistochemistry was performed to investigate whether Robo signalexpresses in T24cell cultured in vitro, and determine the type of this expression.2. Further investigation of immunohistochemistry results were assessed byimmunofluorescence and western blot. 3. Human transitional epithelial cancer cell line of bladder T24was transfectd byRNAi technique through seeking the gene silent sites and constructing a proper expressionvector. Immunofluorescence and western blot were carried out to confirm the RNAi effectsand detect the changes of expression of Robo silencer.4.Survival rate after RNA interfered was evaluated by MTT method.Results1. Robo positive cells were successfully detected by immunohistochemistry andimmunofluorescence in cultured T24. Different antibodie staining showed that expressionsubtypes were Robo1and Robo4, and none of the subtypes is expressed in normaltransitional epithelial cancer cell.2. Western blot results indicated that Robo1and Robo4were found expressed in T24.3. RNAi technique efficiently silence the expression of Robo1and Robo4in T24,especially Robo1, almost completely inhibited (P<0.01).4. Survival rate of Robo1-interfered group, had significant difference (P<0.05) withcontrol group and Robo4group, decreased obviously as showed by MTT method.Conclusions1. Robo1and Robo4protein receptors were demonstrated exist in T24, which provedthat Slit/Robo signal pathway may play an important role in pathologies of bladder cancer.2. Decreasing of expressiong of Robo1and survival rate of tumor cells, afterinterfering of Robo1and Robo4with RNAi technique, indicated that Robo gene may act amajoy part in the growth of bladder tumor, and will potentially become a new gene therapytarget.