An Experimental Study On Twist Gene Expression And RNAi In Bladder Carcinoma

Bladder cancer is the most common tumors of genitourinary tract. The most important clinical character of it is high incidence rate ,high recurrence rate ,and low malignancy. In recent years, molecule mechanisms about bladder cancer is always the hot point, such as gene mutation , protooncogene activation , antioncogene inactivation , apoptosis disequilibrium and so on.The tumor is the result of unlimited cell proliferation, and cell growth is determined by genes.The tumor genes and the tumor suppressor genes exist in the human body, the functions of these two genes are different.The occurrence of tumor is due to increase tumor gene function and inhibit tumor suppressor gene function.A variety of external factors such as radiation, carcinogenic chemicals and viruses induced tumor through the changes of gene.Bladder cancer biological characteristics of malignant include multifocal, multi-gene involvement and heterogeneous multi-stage growth.At present, the treatments of bladder cancer include bladder surgery and anti-cancer drug instillation after surgery.However, easy to relapse after surgery and the height of the chemotherapy drug resistance have been the focus of the study and clinical issues.After nearly 20 years of development,gene therapy has been considered to be following the surgery, chemotherapy and other conventional treatment a very promising way to cure bladder cancer.Twist gene is a novel oncogene that is expressed at higher level in most of the tumors analyzed to date compared to normal tissues. Twist could activate many kinds of tumor related genes, and it has close correlation with tumor generation, development and prognosis. Epithelial mesenchymal transformation( EMT) refers to the epithelial cells into mesenchymal cells in a specific physiological and pathological circumstances.EMT plays important roles in embryonic development, chronic inflammation, tumor progression and a variety of fibrotic diseases.Twist is a important transcription factor in the process of EMT .Twist protein is considered to be a cancer protein, can affect the number of tumor cells apoptosis, invasion and metastasis.Twist maybe a new target in gene therapy.The present study observed the Twist protein expression in human bladder cancer tissues using immunohistochemical methods and analyzed the correlation between Twist protein expression and clinical pathological characters. Using RNA interference technique silencing Twist gene, the effects and its relative mechanism of Twist shRNA on the human bladder cancer cell line T24 cells was observed. The present study was divided into three parts. The follows are:1 Expressions of Twist gene in bladder carcinoma and its clinicopathological significanceObjective: To investigate the expressions of Twist gene in bladder carcinoma and its clinicopathological significance. Methods: By immunohistochemical assay (SP), the expression of Twist gene was done in 5 cases of normal tissues and 95 cases of bladder carcinoma tissues. The results were analyzed by corresponding statistica methods. Results: Twist gene showed higher expression in bladder carcinoma. The rate of positive expression was 87.4% , while the positive rate was 20% in nomal tissues. The difference was of significance between the two tissues (P<0.05). The expressions of Twist gene was correlated with WHO pathology classification and TNM stage (P<0.05), and was incorrelated with age and sex. Conclusions: The expression of Twist is up-regulated in bladder carcinoma tissues , and it plays an important role in progression of tumor.2 Construction and screening of plasmid expression vectors encoding the short hairpin RNA targeting Twist geneObjective: To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting Twist mRNA. Methods: Two plasmid expression vectors coding for shRNA targeting 777 and 845 of Twist gene sequence and a control vector containing random DNA fragment were constructed. The recombinant plasmids were identified by PCR, and then transfected separately into bladder cancer cell line-T24. The Twist gene silencing effect was detected by RT-PCR and Western blotting. Results: The expected band was amplified from the plasmids coding for shRNA by PCR. Transfection of T24 cells expressing Twist gene with the shRNA plasmids resulted in a inhibition of Twist mRNA and protein expressions by 68% and 76%, respectively. Conclusion: The plasmid expression vectors coding for shRNA targeting Twist mRNA have been constructed successfully,of which pGenesil-shRNA1 most effectively silences Twist gene in T24 cells.3 Effect of silencing Twist gene expression by RNA interference on proliferation of human bladder carcinoma T24 cell lineObjective: To investigate whether Twist gene downregulation by RNA interference (RNAi) leads to inhibition of proliferation and arrest of cell cycle in human bladder carcinoma T24 cell line. Methods: The shRNA expression plasmid targeting to Twist gene was constructed and transfected into bladder cancer T24 cell line with Lipofectamin2000. RT-PCR and Western Blot was used to monitor the validity of specific shRNA in downregulation of Twist expression. Then the MTT assay was performed for detecting cell proliferation and PI flow cytometric analysis for cell cycle. Results: The specific Twist shRNA was confirmed to be efficient in silencing Twist expression. Twist gene downregulation by RNAi inhibited cell proliferation remarkably( P < 0. 05) and arrested cell cycle at G1 phase significantly( P < 0. 05). There was an increase of cell number at G1 phase [ (74.34±3.24) % vs (48. 17±1.62) % , P < 0. 05 ] and a decrease of S phase [ (11.58±1.02) % vs (33.71±3.54) % , P < 0. 05 ] in T24 cells treated with Twist shRNA compared with untreated T24 cells. Conclusions: Twist specific shRNA expression vector could specifically and efficiently inhibit the expression of Twist gene to regulate cell cycle and inhibit cell proliferation in human bladder carcinoma T24 cell line.In summary, Twist gene expression products might be the auxiliary diagnosis way in assessing bladder cancer malignance and invasion ability, which might direct the bladder cancer operation and further treatment. The present study showed that Twist shRNA plasmids could silence Twist gene effectively and RNAi gene therapy targeting Twist might offer the new direction for treatment bladder cancer.