Dissertation
Dissertation > Medicine, health > Oncology > Sports Department tumor > Bone tumor

The Effect and Mechanism of Twist on Metastasis and Epithelial Mesenchymal Transition of Osteosarcoma

Author YinKe
Tutor LiaoQianDe
School Central South University
Course Clinical
Keywords Twist osteosarcoma epithelial mesenchymal transition E-cadherin N-E-cadherin β-Catenin metastasis survival rate
CLC R738.1
Type PhD thesis
Year 2013
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Objective1. To investigate the expression of Twist in human osteosarcoma and osteofibrous dysplasia, and to analyse the relationship between the Twist gene expression and the clinical features of osteosarcoma.2. To analyse the expression of Twist and E-cadherin in human osteosarcoma; and to analyse the effect of Twist and E-cadherin expression on the prognosis and the survival rate of patients with osteosarcoma.3. To analyse the influences of E-cadherin, N-cadherin,β-catenin, which proteins associated with EMT, because of silencing Twist gene on osteosarcoma cells MG-63.4. To analyze the biological characteristics of the osteosarcoma cells MG-63after silencing Twist gene.Methods1. The Twist protein expression in the107osteosarcoma tissue specimens and the25osteofibrous dysplasia tissue specimens was detected by immunohistochemistry. The relationship was statistically analyzed between the Twist protein expression and the clinicopathological features, such as age, gender, tumor size, tumor location, histological subtype, recurrence and metastasis.2. The E-cadherin protein expression in the107osteosarcoma tissue specimens were detected by immunohistochemistry. The relationship was statistically analyzed between the co-expressions of Twist and E-cadherin and the survival rate of patients with osteosarcoma. Risk of survival rate in patients with osteosarcoma was analyzed using multiple Cox regression analysis.3. The specific Twist siRNA was transfected into the osteosarcoma cells MG-63using lipofectamine. The most efficient siRNA was screened out, according to the expression of Twist mRNA and protein detected by the real-time PCR and the western blot method. After transfecting the best siRNA to silence Twist expression in the osteosarcoma cells MG-63, the real-time PCR and the western blot method were performed to detect the different expression of mRNA and protein in E-cadherin, N-cadherin, and β-catenin in different groups.4. After transfecting the best siRNA to silence Twist expression in the osteosarcoma cells MG-63, the cell proliferation was measured by MTT assay; the cell cycle and cell apoptosis were detected by flow cytometry; and the Transwell assay analysis was done to measure the invasion and migration of the cells.Results1. The positive expression rate of Twist in osteosarcoma tissue(31.8%) was significantly higher than that in osteofibrous dysplasia tissue(8%)(X2=5.776, P<0.05).The positive expression rate of Twist in osteosarcoma cases with metastasis was significantly higher than that in osteosarcoma cases without(52.8%VS11.1%,X2=21.474, P<0.05).2. The positive expression rate of E-cadherin is20.6%(22/107) in osteosarcoma tissue specimens. The positive expression rate of E-cadherin in osteosarcoma cases with metastasis was significantly lower than that in osteosarcoma cases without(X2=21.474, P<0.05). A significantly negative correlation was found between the expression of E-cadherin and the expression of Twist in the osteosarcoma tissue (rs=-0.209, P<0.05). A positive expression of Twist expression in osteosarcoma was correlated with a shorter disease-free survival and overall survival (respectively, both P<0.05), while a positive expression of E-cadherin expression was correlated with a shorter overall survival (P<0.05), but in the disease-free survival, a positive expression of E-cadherin were not (P>0.05). The expression of Twist is independent risk factor for overall survival rate and disease survival in patients of osteosarcoma (P<0.05).3. The Twist siRNA#806group is the most effective group in the three Twist siRNA groups. On the expression of mRNA and protein in E-cadherin, the Twist siRNA#806group was lower than the Twist siRNA#NC group and the blank group respectively (both, P<0.05), while the Twist siRNA#806group was higher than the Twist siRNA#NC group and the blank group respectively on the expression of mRNA and protein in N-cadherin and β-catenin respectively (both, P<0.05). In the12h,24h,48h,72h MTT results showed that the cell proliferation of the Twist siRNA#806group was significantly lower than that of the Twist siRNA#NC group and the blank group respectively (both, P<0.05). The flow cytomety demonstrated that the cell proportion in sub G0/G1phase of the Twist siRNA#806group was higher than that of the Twist siRNA#NC group and the blank group respectively (both, P<0.05). The flow cytomety demonstrated that the percentage of apoptosis cells in the Twist siRNA#806group was higher than that in the Twist siRNA#NC group and the blank group respectively (both, P<0.05). Transwell cell migration and invasion experiment results show that the migration and invasion cell number in the Twist SiRNA#806group was significantly lower than that in the Twist SiRNA#NC group and the blank group respectively (both, P<0.05). Conclusion1. The over expression of Twist gene in osteosarcoma tissue was correlated with the metastasis of osteosarcoma, may become the early diagnosis marker for osteosarcoma.2. Twist and E-cadherin might be related to the prediction of metastasis potency and poor prognosis for patients with osteosarcoma, and that Twist was an independent risk factor for prognosis of patients with osteosarcoma.3. Twist played an important role in the EMT process of osteosarcoma and contribute to tumor metastasis, and might become a potential target for the specific gene treatment to osteosarcoma probably.

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