Study On The Relationship Between The Twist Gene And The Invasion, Metastasis And Epithelial-mesenchymal Transitions Of Ovarian Cancer

Background Metastasis is a complex multi-step process.Ovarian cancer is prone to invade and disseminate in abdominopelvic cavity, therefore, how to inhibit cancer cell invasion and metastasis become the central issue in clinical treatment and basic research of ovarian cancer. Epithelial-mesenchymal transitions (EMT), during which epithelial cells transform from the polary epithelial phenotype to a high degree of movement of the fibroblast-like or mesenchymal phenotype, play an important role in early embryogenesis. In vitro and in vivo studies show that, EMT play an important role in the infiltration and dissemination of epithelial tumours. EMT which are activated during the progress,infiltration and invasion of epithelial tumour, are key molecular events of invasiveness acquirement of epithelial tumor cells, and play a major pathological role in the development and metastasis of malignant tumors spread process. As to the Biochemistry, cell changes the expression of epithelial markers (such as E-cadherin) to the expression mesenchymal markers (such as N-cadherin). Originally found in the study of embryonic development of Drosophila shows that, the Twist gene could regulate cell migration and tissue remodeling, and induct the mesoderm development. The current studies have confirmed that, Twist gene is closely related to the EMT phenomenon and neoplasm metastasis, also is the key regulatory factor of EMT progress. At present, there is no intensive study in the relationship beteen Twist gene and EMT, invasion and metastasis of ovarian cancer. We studied the role of Twist gene in the happenness,development and EMT,metastasis of ovarian cancer. Our study is devided into two parts.Part OneObjective By detecting the protein and mRNA expressions of Twist, E-cadherin, of N-cadherin in ovarian cancer tissues to investigate the roles of Twist gene in the happenness, development, and EMT metastasis of ovarian cancer.Method1. Use immunohistochemical method to detect the expressions of Twist, E-cadherin and N-cadherin protein in54cases of ovarian cancer tissues and54cases normal ovarian tissue3, and compare the expressions of the three indicators in ovarian cancer tissue and normal ovarian tissue. Analyze the expressions of Twist, E-cadherin and N-cadherin protein and the correlation with stage, differentiation and lymph node metastasis of ovarian cancer.Analyze the correlation between the expreesions of E-cadherin and Twist protein in ovarian cancer, and the correlation between the expreesions of N-cadherin and Twist protein in ovarian cancer.2. Apply RT-PCR method to detect the expressions of Twist, E-cadherin and N-cadherin mRNA in54cases of ovarian cancer tissues and54cases of normal ovarian tissue. Compare the expressions of Twist, E-cadherin, N-cadherin mRNA in ovarian cancer tissue and normal ovarian tissue.Results1. In the ovarian cancer tissue, the positive expression rate of Twist, E-cadherin and N-cadherin protein were68.5%,27.8%and48.1%respectively, while in normal ovarian tissue, they were25.9%,82.5%and14.8%respectively.The differences of the three indicators between ovarian cancer tissue and normal ovarian tissue had statistical significance (P<0,05).2. The expression of Twist in the ovarian cancer tissue with high grade, late stage and with lymph node metastasis were higher than that of low grade, early stage and without metastasis of lymph node.The expression of E-cadherin in the ovarian cancer tissue with high grade, late stage and with lymph node metastasis were lower than that of low grade, early stage and without metastasis of lymph node.The expression of N-cadherin in the ovarian cancer tissue with high grade, late stage and with lymph node metastasis were higher than that of low grade, early stage and without metastasis of lymph node.Tthe differences all had statistical significance (P<0,05).3. The relative abundance of Twist, E-cadherin and N-cadherin mRNA in the ovarian cancer tissue (optical density ratio)were1.49±0.53,0.82±0.24,1.55±0.56respectively. While in normal ovarian tissue, they were1.14±0.38,1.08±0.19,1.14±0.32respectively. The differences of all the indexes above between the expression in ovarian cancer tissue and normal tissue had statistical significance (P<0,05).4. Among37ovarian cancer tissues with positively expressed Twist,33of them lack E-cadherin expression; while among17ovarian cancer tissues with negative expressed Twist,6of them lack E-cadherin expression.The expression of E-cadherin negativly correlated with the expression of Twist in ovarian tissue, r=-0.56. Among37ovarian cancer tissues with positively expressed Twist,23of them positivly expressed N-cadherin; while among17ovarian cancer tissues with negatively expressed Twist,3of them positively expressed N-cadherin.The expression of N-cadherin positively correlated with the expression of Twist in ovarian tissue, r=0.41.Conclusions1.The expression of the Twist protein and mRNA was upregulated in ovarian cancer tissues, suggesting that Twist gene seemed to correlate with the happening of ovarain cancer.2. When the Twist expression in the ovarian cancer tissue was up-regulated, the epithelial marker E-cadherin expression is down-regulated and meanwhile the mesenchymal marker N-cadherin expression was up-regulated, suggesting that Twist presumably correlate with EMT in ovarian cancer.3.The expression of the Twist and N-cadherin were upregulated while the expression of E-cadherin was downregulated in the late stage, high grade, and lymph node metastasis of ovarian cancer, which suggested that Twist and epithelial-mesenchymal conversion which was possibly relevant to Twist may be related to the development of ovarian cancer. Object Apply RNAinterference (RNAi) technique to inhibit ovarian cancer A2780cell to express Twist.Observe the changes of proliferation, invasion, adhesion properties of ovarian cancer A2780cell in vitro, and the changes of expression of E-cadherin and N-cadherin after Twist gene silence.Method1. pGenes12eukaryotic expression vector to was used to construct two pairs of recombinant pGenesil2-Twist shRNAl and pGenesil2-Twist shRNA2which contain efficient targeting Twist gene. Then the recombinant vectors were identified by enzyme digestion and gene sequencing.2. pGenesil-2Twist shRNA plasmid was transfected into human ovarian cancer A2780cells. After transfection at24h,48h and72h cells were collected, then fluorescence microscopy was used to measure and observe transfection efficiency and transfection conditions.3.Western-blot was used to detect the expression of Twist protein respectively in A2780-nontransfection, A2780-si-control(A2780cells transfected with control siRNA vecto expressing siRNA that does not match any known human coding mRNA),and A2780-Twist-shRNA group cells, and analyze the changes of Twist protein expression in human ovarian cancer A2780cell after transfected with pGenesil-2Twist shRNA plasmid.4.Twist mRNA expression of A2780-nontransfection,A2780-si-control and A2780-Twist-shRNA group cells was detectd by RT-PCR.5.The proliferation of A2780-nontransfection,A2780-si-control and A2780-Twist-shRNA group cells was examined by MTT method.6.The invasion ability in A2780-nontransfection, A2780-si-control and A2780-Twist-shRNA group cells was examined by Transwell chambers.7. The adheison assay was used to detect the cancer cells of adheison ability.8. RT-PCR method was applied to detect the mRNA expression of E-eadherin and the N-cadherin of ovarian cancer cells in A2780-nontransfection,A2780-si-control and A2780-Twist-shRNA group, and analyze the influence on the mRNA expressions of E-eadherin and N-cadherin with pGenesil-2Twist shRNA plasmid transfection.9. Westemblot was used to detect the protein expression of E-eadherin and N-cadherin of ovarian cancer cells in A2780-nontransfection,A2780-si-control and A2780-Twist-shRNA group.Results1.Enzyme digestion and gene sequencing revealed that restructuring pGenesil2-Twist shRNA1and pGenesi12-Twist shRNA2had been successfully constructed.2.Green fluorescence(GFP) could be observed in the successfully transfected cells through a microscope.The transfection rate of the ovarian cancer A2780cell after transfected Twist shRNA plasmid24h was above70%, and the transfection rate after transfected48h was above80%.3. RT-PCR determine? the Twist mRNA expression after transfected pGenesil2-Twist shRNA plasmid48h showed that, the ratio of amplification product of Twist (455bp) to GAPDH in A2780-nontransfection,A2780-si-control,Twist-shRNA1-A2780group and Twist-shRNA2-A2780group cells were (1.04±0.028,1.10±0.026,0.38±0.033and0.51±0.029) respectively. There was no statistically difference between A2780-nontransfection group and A2780-si-control group cells(P>0.05), while Twist-shRNAl-A2780group and Twist-shRNA2-A2780group showed significantly lower than A2780-nontransfection and A2780-si-control group(P<0.05). The interference efficiency of transfection of Twist shRNAl plasmid wsa65%, and the interference efficiency of transfection of Twist shRNA2plasmid was56%.4. Western blot test showed that the Twist protein expression in A2780-nontransfection and A2780-si-control group were significntly higher than that Twist-shRNA1-A2780group and Twist-shRNA2-A2780group(P<0.05). The expression of Twist protein in Twist-shRNA1-A2780group was the lowest,in accordance with the result of RT-PCR test.Thus we selected the shRNA1plasmid to do subsequent experiments.5. MTT assyay showed that, after24h,48h, and72h of the transfection, there is no significant difference in the proliferration among A2780-nontransfection,A2780-si-control and A2780-Twist-shRNAgroup (P>0.05).6Transwell chambers experiment showed that, compare with A2780-nontransfection and A2780-si-control group, the invasion cells in A2780-Twist-shRNAgroup decreased significantly (P<0.05). The number of invasion cells in A2780-nontransfection and A2780-si-control group had no statistical difference (P>0.05).7. Adhesion assay showed that, comparing with A2780-nontransfection and A2780-si-control group, the adhesion capacity of the cells in A2780-Twist-shRNAgroup decreased significantly (*P<0.05).8. RT-PCR and Western-blot assay showed that the mRAN and protein expression of E-cadherin increased significantly in??or of A2780-Twist-shRNAgroup compare with that of A2780-nontransfection and A2780-si-control group, while the expression of N-cadherin decreased significantly compared with that of A2780-nontransfection and A2780-si-control group (P<0.05).there is no significant difference in the expression of E-cadherin and N-cadherin between A2780-Twist-shRNA group and A2780-si-control group(P>0.05).Conclusion1. Restraining the expression of Twist gene in the human ovarian cancer A2780cell did not influence the reproductive activity of ovarian cancer cells in vitro, but it could decreased the invasion and adhesion ability of ovarian cancer cells in vitro.2. Restraining the expression of Twist gene in the human ovarian cancer A2780cell could restrain epithelium-mesenchymal conversion in the ovarian cancer cells.3.In the process of epithelium-mesenchymal conversion and invasion and metastasis of the ovarian cancer, Twist probably plays an important role.Restraining Twist expression may become a new target of ovarian cancer treatment.