| Bladder cancer is one of the most common malignant tumors in China, the incidence rate among 7 of the World tumor, the most common cancer of the bladder from urothelial carcinoma.The development of bladder cancer is a process of multi-gene participation, multi-stage and a variety of factors regulating, Therefore, in-depth study the gene regulation mechanism of bladder cancer and explore the effective treatment measures are the priorities and difficulties in diagnosis and treatment of bladder cancer, and there is have a great significance of prevention and treatment in bladder cancer.Eukaryotic initiation factor 4E (eIF4E) is an important regulatory factor of eukaryotic protein synthesis, and is play a very important role in protein synthesis in the initial stage. Studies have shown that, the high expression of eIF4E is closely related to the occurrence of a variety of tumor. In recent years, there have been reported in the literature the high expression of eIF4E or increased activity is closely related to the occurrence of head and neck squamous cell carcinoma, cervical cancer, breast cancer, esophageal cancer, colon cancer and other tumor. The expression of eIF4E in invasive bladder urothelial carcinoma and its relationship with the tumor occurrence and development there is no systematic study reported so far at home and abroad.For further research on the relationship between eIF4E and invasive bladder urothelial carcinoma and find the effective way to inhibit the occurrence and development of bladder invasive urothelial carcinoma, in this study, using immunohistochemistry and in situ hybridization combined detect the expression of protein and mRNA levels of eIF4E and associated factors of heparanase(HPA) in bladder invasive urothelial carcinoma, bladder papilloma and normal bladder mucosa. On this basis, use liposome-mediated method transfected three eIF4E antisense oligonucleotides (ASODN) sequence in bladder cancer BIU-87 cells with different concentrations and different time. Then, use immunocytochemistry, Western blot, in situ hybridization and RT-PCR methods detect the protein and mRNA expression of eIF4E and HPA in each group of BIU-87 cells; Detect each group of cell growth inhibition rate by MTT; Use TUNEL and flow cytometry to detect the apoptotic cells in each group, And filter out the best transfection sequence. Finally, by the experiments of xenografts in nude mice, detect the expression of eIF4E gene in the bladder transplanted tumors of nude mice by RT-PCR, in situ hybridization and immunohistochemical, respectively Use the immunohistochemistry and in situ hybridization detect the eIF4E protein and mRNA expression in bladder cancer xenografts; and use TUNEL detect the apoptosis of bladder cancer xenograft cells. Observe the role of the eIF4E gene on xenografts in nude mice in vivo, Attempt to clarify the significance of eIF4E expression in invasive bladder urothelial carcinoma and gastric cancer occur and development. In order to provide a theoretical basis to the targeted therapy of bladder cancer. This study is divided into three parts.Part I Expressions of eIF4E and HPA in bladder invasive urothelial carcinoma tissues and their significanceMethods1. Using immunohistochemistry and in situ hybridization to detect the expression of eIF4E gene in 50 cases of bladder invasive urothelial carcinoma,30 cases of bladder papilloma and 50 cases of normal bladder mucosa.2. Using immunohistochemistry and in situ hybridization to detect the expression of HPA gene in 50 cases of bladder invasive urothelial carcinoma,30 cases of bladder papilloma and 50 cases of normal bladder mucosa3. Software of SPSS 13.0 was used to analysis the statistics, test standardα= 0.05, chi-square test,T test and variance analysis and Spearmanm were used.Results1. Expression of the eIF4E and HPA gene was lower or no in bladder papilloma and normal bladder mucosa membrane tissue compared to BIUC. Both the expression of eIF4E, HPA protein and mRNA were mainly detected in in cytoplast of BIUC.2. Increased expression levels of protein and mRNA of eIF4E were seen from normal bladder mucosa membrane tissue, bladder papilloma tissue to BIUC tissue, and there were significant differences among three groups (P<0.05).3. Gradually increased levels of the expression of HPA protein and mRNA were observed from normal bladder mucosa membrane tissue, bladder papilloma tissue to BIUC tissue, and there was significant difference among three groups (P< 0.05)4. The expression of eIF4E protein and mRNA was positively correlated with the expression of HPA protein and mRNA(r=0.504,P<0.05)0Part II The influence of eIF4E ASODN to the expression of eIF4E and HPA in human bladder cancer BIU-87 cellsMethods1. Use liposome-mediated method to transfect eIF4E ASODN into bladder cancer BIU-87 cells with 2.5,5.0 and 7.5μg/ml for 24,48 and 72h, respectively. And set up cells control group (not transfected), blank control group (transfected with empty liposomes) and unrelated control group (transfected with unrelated control ASODN).2. Use immunocytochemistry, Western blot, in situ hybridization and RT-PCR to detecte the expression of eIF4E and HPA protein and mRNA in each group BIU-87 cells.3. Detect each group of cell growth inhibition rate by MTT.4. Use TUNEL and flow cytometry to detect the apoptotic cells in each group.5. Statistics analysis was performed by software of SPSS13.0, the standard of test:α=0.05, chi-square test and one-way analysis of variance were used. Results1. The expression of eIF4E and HPA were decreased in each eIF4E ASODN transfection group cells compared with control group. In eIF4E ASODN transfection group, there were significant differences compared with cells control group, blank control group and unrelated control group (P<0.05),which have time and concentration dependence.2. The cell growth inhibition rate of eIF4E ASODN transfection group significantly higher than cells control group,blank control group and unrelated control group (P<0.05). The highest inhibitory rate of 7.5μg/ml transfected 72 hours3. There was significant difference of cell apoptosis rate between eIF4E ASODN transfection group and each control groups. And the most obvious is 7.5μg/ml transfected 72 hours.Part III Influence of eIF4E ASODN on the growth of xenografts in nude miceMethods1. Transplantable tumors were produced using BIU-87 cells and the transfected BIU-87 cells with eIF4E ASODN transfection group, unrelated control group and untransfected group, respectively, in nude mice. Compare the tumorigenicity and the tumor size in each group of nude mice2. Detect the expression of eIF4E gene in the bladder transplanted tumors of nude mice by RT-PCR, in situ hybridization and immunohistochemical, respectively.3. Use the immunohistochemistry and in situ hybridization detect the eIF4E protein and mRNA expression in bladder cancer xenografts.4. Use TUNEL detect the apoptosis of bladder cancer xenograft cells.5. Statistical analysis:Statistics analysis was used by SPSS13.0 software,usingchi-square test,T test and variance analysis,test standard a=0.05.Results1. The volume of xenografts in nude mice of unrelated control group and untransfected group was significantly greater than eIF4E ASODN transfection group. Statistically significant difference between groups (P<0.05)2. The expression of eIF4E protein and mRNA were Significantly higher in unrelated control group, untransfected group and bladder cancer cell group compare with eIF4E ASODN transfection group. Statistically significant difference between groups (P<0.05)3. The TUNEL method detected apoptotic cells of bladder cancer xenograft was show that the nucleus was tan, dyed shades vary, chromatin uneven distribution. Apoptotic index in unrelated control group and untransfected group was no significant difference(P> 0.05), the two groups of apoptotic index was significantly lower than the eIF4E ASODN transfection group, the differences were significant (P<0.05),Conclusions1. High levels of eIF4E protein and mRNA were observed in bladder invasive urothelial carcinoma,suggesting that eIF4E may be associated with the occurrence and development of bladder invasive urothelial cancer.2. High expression of eIF4E gene can up-regulate the expression of HPA, thus contributing to tumor invasion。3. The best transfection sequence of eIF4E ASODN was successfully screened, and introduced into BIU-87 cells. BIU-87 eIF4E gene inhibition cells were successfully established,which will lay a foundation for further studing the biological function of eIF4E and eIF4E target therapy.4. eIF4E ASODN can reduce the expression of eIF4E in the BIU-87 cells in vitro, and down-regulate the expression of HPA protein.5. eIF4E ASODN obviously inhibites the growth of transplantable tumors and promote apoptosis of transplanted tumor cells in vivo, which will provide the new idea and theoretical basis for target gene therapy of BIUC.
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