The Study Of The Biological Effects Of TRPV2on Bladder Tumor Cells

In China, bladder carcinoma is the most common malignancy of urinary tract, and the most common type of urothelial tumor diagnosed is transitional cell carcinoma, and it is the important causes contributed to the human death. Limitations of bladder tumors are relatively limited threat to human health, but the nutrient consumption caused by the rapid growth of bladder tumors, the local invasion and distant migration of bladder tumors led to the death of the patient. However, the current treatment of bladder cancer is based on the surgical treatment mainly assisted radiotherapy, chemotherapy and immunity modulation therapy, but the treatment effect is not satisfactory. Looking for new treatments become the new way for the prevention and treatment of bladder tumors, gene therapy has become one of hot area research. TRPV2ion channel is confirmed may be related to the development and progression of bladder cancer, The purpose of this experiment is to investigate the impact of TRPV2bladder tumor cell lines in vitro, and to explore the possibility of TRPV2as the bladder cancer treatment target gene.This experiment was divided into three parts:[1] The expression of TRPV2in the bladder cancer5637, T24, EJ cell lines;[2] The construction and identification of the eukaryotic expression vector encoding protein of rat transient receptor potential V2;[3] The role of cationic channel TRPV2on proliferation, migration and invasion of bladder cancer5637and EJ cells.Part I:The expression of TRPV2in the bladder cancer5637, T24, EJ cell linesObjective:To detect the expression of TRPV2in bladder cancer cell line5637, T24, and EJ cells.Methods:Conventional cell culture, extracted the total RNA and protein from bladder tumor cell lines5637T24EJ cells. TRPV2mRNA and protein levels were detected by RT-PCR and western blot.. Results:The expected bands were detected in the destination location in all5637, T24, the EJ cell lines, and transcriptional and translational levels were consistent luminance strip. The strip luminance values of T24of EJ cells were significantly higher than the luminance value of5637cells.Conclusions:TRPV2mRNA and protein levels were all detected in bladder carcinoma cell line5637, T24and EJ cells. The level of TRPV2in malignant degrees higher and poorly differentiated bladder tumor cell lines was higher, while the expression in the relatively better differentiation of low malignant bladder tumor cell lines was lower. Part Ⅱ:The construction and identification of the eukaryotic expression vector encoding protein of rat transient receptor potential V2Objective:To construct and identify of the eukaryotic expression vector encoding protein of rat transient receptor potential V2.Methods:The primer of the full-length target sequence was designed by according to the rat-derived gene library in the GeneBank database provided of TRPV2the mRNA sequence. PCR was used to amplify the target gene fragments. The recombinant plasmid was constructed by double digestion product connected to the pcDNA3.1+plasmid. The sequence of the recombinant plasmid was blasted with the sequences from the GeneBank.Results:The expected size gene fragment was obtained from PCR and digestion. Gene sequencing showed the nucleotide sequence of recombinant plasmids was consistent with the the nucleotide sequence from the GeneBank.Conclusion:the eukaryotic expression vector encoding protein of rat transient receptor potential V2was constructed successfully. Part III:The role of cationic channel TRPV2on proliferation, migration and invasion of bladder cancer5637and EJ cellsObjective:To investigate the role of cationic channel TRPV2on proliferation, migration and invasion of bladder cancer5637and EJ cells.Methods:We transfected with a cDNA encoding the rat TRPV2protein to5637cells, and transfected with a sh-RNA-TRPV2to EJ cells. These two types of cells were divided into three groups, the target fragment transfected group, the empty plasmid transfected group, the normal cells group. RT-PCR and western blot were used to assay the expression of the transfected plasmid. Cell proliferation abilities were assayed by flow cytometry (FCM) and MTT assay. Cell migration abilities were assayed by the scratch assay analysis and transwell assay.Results:The results of the RT-PCR and the western blot prompted the target fragment gene has been transfected to the5637and EJ cells. Cell cycle distribution and cell growth curve revealed that the over-expression of TRPV2had no effect on the cells multiplication rate (P>0.05) in5637cells in vitro, while the sh-RNA-TRPV2could inhibit the proliferation and growth of the EJ cell in vitro, and the results of scratch assay, transwell assay suggest that TRPV2enhances the cell invasion ability in vitro.Conclusion:These results suggest that the over-expression of nonselective cationic TRPV2channel enhanced bladder cancer5637cells migration but no effect on the cell proliferation in vitro, and sh-RNA-TRPV2could inhibit the growth, proliferation, migration and invasion abilities of EJ cells. TRPV2may be a new target for therapy of bladder tumors.