The Research Of The Effects Of Recombined Lentviral CXCR7-shRNA On Biological Behavior Of The Human Bladder Cancer Cell

Chapter1The expression of chemokine receptor CXCR7in human bladder cancerObjective To determines the expression of CXCR.7in the human bladder cancer tissue,and in the human bladder cancer cell lines and choose high expression level one into the next experiment.Methods1. Determine the CXCR7expression in the human bladder cancer tissue, the bladder tissue closing to the carcinoma and the non-carcinoma bladder tissue respectively by using the immunohistochemistry method. And find out the relationship between CXCR7expressing level and the pathological grade and stage of the bladder cancer.2. Determine the CXCR7-mRNA expression in the human bladder cancer cell line5637and T24by using QPCR.Results1. There was higher CXCR7expression in bladder cancer than in closing carcinoma tissue and non-carcinoma bladder tissue (p<0.05), but there was no difference between closing carcinoma tissue and non-carcinoma bladder tissue of the CXCR7expression (p=0.55).2. There was significance relationship between CXCR7expression of bladder cancer and pathological grade (p=0.011) and stage (p<0.05).3. The expression of CXCR7-mRNA was more higher in5637cell line than T24cell line.Conclusion1. The CXCR7was expressed in the bladder cancer but low expression or non-expression in closing carcinoma tissue and non-carcinoma tissue and there was a significance relationship between CXCR7expression level and pathological grade and stage in bladder cancer.2. The5627cell line was chosen to the next experiments because the high expression level of CXCR7-mRNA. Chapter2The construction of lentviral vector intermediate CXCR7-shRNAObjective To screen the effective CXCR7-siRNA sequence and construct the LV-CXCR7-shRNAMethods1.To construct the pSilencerTM4.1-CXCR7-shRNA-1,2,3and pcDNA3.1(+)-CXCR7by using the Designed CXCR7-siRNA sequence and CXCR7sequence respectively. Make the pSilencerTM4.1-CXCR7-shRNA and pcDNA3.1(+)-CXCR7to transfect the293T cell together. Screen the effective CXCR7-siRNA sequence by QPCR and western blot.2. Use the three plasmids lentivirus vector system, which made by Invitrogen, to construct LV-CXCR-shRNA based on the effective CXCR7-siRNA sequence for preparing the next experimentsResults After transfecting the pSilencerTM4.1-CXCR7-shRNA-1,2,3and pcDNA3.1(+)-CXCR7to293T cell together the expression of CXCR7-mRNA and protein was dropped (p<0.05). The CXCR7-mRNA was down expressed3.0%,91.54%and89.06%by CXCR7-siRNA-1,2,3respectively.2. There was no variation and insert of the construction of LV-CXCR7-shRNA based on CXCR7-siRNA-1in the sequence test. The results show that the LV-CXCR7-shRNA was completely and successfully built. The titer of LV-CXCR7-shRNA was4.2x108and fulfilled the next experiment.Conclusion CXCR7-siRNA-1was the most effective interference sequence and the LV-CXCR7-shRNA was successfully constructed based on CXCR7-siRNA-1 Chapter3The effect of LV-CXR7-shRNA on biological behavior in human bladder cancer cell5637cell lineObjective Determine the interference effect on5637cell line by LV-CXCR7-shRNAMethods Make three groups of the cultured5637cell line and transfect the LV-CXCR7-shRNA plasmid, LV-shRNA negative control plasmid and blank lentviral plasmid to the three grouping5637cell lines respectively. Determine the expression of CXCR7-mRNA and protein by QPCR and western blot respectively in each grouping cell lines. Determine the capability of cell multiplication in each grouping cell lines by MTT experiment. Determine the invasion ability of each grouping cell lines by Transwll experiment.Results Three different of plasmids were successfully transfected to the three grouping5637cell lines. Compare to Con group and NC group the expression of CXCR7-mRNA and protein was dropped in shRNA group (LV-CXCR7-shRNA)(p<0.05). The MTT experiment shows that the capability of cell multiplication in shRNA group was descended than the cell lines in Con group and NC group (p<0.05). The invasion ability of cell line in shRNA group was descended than the cell lines in Con group and NC group by the Transwell experiment (p<0.05).Conclusion There was a relationship between CXCR7and the capability of cell multiplication and invasion ability of human bladder cancer5637cell line. The LV-CXCR7-shRNA can descend the capability of cell multiplication and invasion ability and promote the apoptosis of5637cell lines.