| Objective: Choriocarcinoma is short for CC and it has the highest degree of malignancy in gestational trophoblastic diseases(GTD). It can secondary to any types of pregnancy, including hydatidiform mole, spontaneous abortion, ectopic pregnancy or term delivery. The incidence of choriocarcinoma in female reproductive system tumors is lower than cervical cancer, ovarian cancer and endometrial cancer, but it developed rapidly and can early metastasis to many important organs such as lung, brain etc, so it often endangers women’s health and life seriously. Because of the extremely sensitive to chemotherapy drugs, choriocarcinoma becomes the first gynecological solid tumor that can be cured by chemotherapy. However, because the choices of the chemotherapy drugs and chemotherapies were undeserved, drug resistance in tumor cells often occurs, this becomes the main reason for the failure of chemotherapy. The incidence of gestational trophoblastic tumor is associated with multiple factors and its pathogenesis is not clear, so the research on its occurrence, development and drug resistance has been widely concerned. It has been proved that epithelial-mesenchymal transition(EMT) plays an important role in the local invasion and distant metastasis in a variety of tumors. Twist is one of the members of b HLH proteins family and was first discovered in the study of drosophila embryonic development by Thisse in 1983.The overexpression of Twist are common in metastatic tumors. In the study of all kinds of tumors we found that Twist gene is one of the main factor that can induce epithelial-mesenchymal transition(EMT) and get the migration, invasion and metastasis ability on tumor cells. This study is to investigate the overexpression of Twist gene in human choriocarcinoma JEG-3 cells and its influence to the epithelial cell marker E-cadherin, interstitial cell marker N-cadherin and the proliferation ability of choriocarcinoma JEG-3 cells. Explain the role of Twist gene in the occurrence and development in choriocarcinoma, provide another way to illustrate the pathogenesis of choriocarcinoma and more reliable theoretical basis for choriocarcinoma’s early diagnosis and treatment, so that develop an individual treatment plan.Methods:1 Culture of cells: Cultivate choriocarcinoma cell line of JEG-3 in the incubator of 37℃,5%CO2 to keep a good state.2 Group:(1)the experimental group-transfected with p EGFP-N1-Twist specific plasmid;(2)the negative control group-transfected with the empty plasmid;(3)the liposome control group-transfected with LipofectamineTM 2000 liposome transfection reagent;(4)the blank control group-normal culture without transfection.3 Inverted fluorescence microscope was used to observe the morphological changes after transfection.4 The cellular growth rate of each group cells was assayed by CCK-8 at the time of 0h,24 h,48h,72 h,and 96 h.5 The Twist-m RNA expression in the choriocarcinoma cells after transfection 48 h was detected by Realtime-q PCR.6 The E-cadherin and N-cadherin expression in the choriocarcinoma cells after transfection 48 h was measured by Western blot analysis.7 Statistical method: Data were evaluated using SPSS 13.0 statistical software, and data were recorded as `c±s. Means of multiple groups were analyzed using single factor analysis of variance, differences between groups using SNK/LSD methods. P<0.05 was considered as a significant difference. All the experimental results repeated 3 times.Results:1 The morphological change observation: The JEG-3 cells were round or polygonal shaped with a clear outline,48 h after transfection, the cell morphology were changed to irregular and grown as a large patch, the cell outline were unclear.2 The CCK-8 experiment:0 hour, The proliferation of the experimental group(0.143±0.003),the negative control group(0.138±0.003),the liposome control group(0.143 ± 0.003)and the blank control group(0.139 ± 0.002), compared between each groups, P>0.05, there has no significant statistically differences. The proliferation were similar;24 hours: The proliferation of the experimental group(0.248 ±0.002)compared with the negative control group(0.244±0.007), P>0.05, no significant statistically difference. The proliferation were similar; the experimental group(0.248±0.002) compared with the liposome control group(0.193±0.004)and the blank control group(0.221±0.010), P<0.05,the difference was statistically significant,that means the experimental group has the faster proliferation.48 hours: Compare between the negative control group(0.417±0.003), the liposome control group(0.407±0.011), the blank control group(0.418±0.009), P>0.05, no significant statistically difference between them, they have the similar proliferation rate. However, the experimental group(0.515±0.002)compared with the other 3 control groups, P<0.05,the difference was statistically significant, proliferation was obvious faster.72 hours: The cells proliferation rate were the experimental group(0.988±0.021)>the blank control group(0.781±0.016)>the negative control group(0.617±0.003)>the liposome control group(0.515±0.013), P<0.05,the difference was statistically significant;96 hours: The cells proliferation of each group were similar to 72 hours, the experimental group(1.411±0.005)>the blank control group(1.293±0.003)>the negative control group(1.163±0.006)>the liposome control group(0.801±0.005), P<0.05,the difference was statistically significant.3 The Realtime-q PCR test: To detect the Twist-m RNA expression in the choriocarcinoma cells after transfection 48h: the experimental group(2.074±0.025); the negative control group(1.093 ± 0.011); the liposome control group(1.055 ± 0.025); the blank control group(1.002 ± 0.021). The experimental group compared with the other 3 groups, P<0.05, the difference was statistically significant. That indicate the Twist-m RNA expression of experimental group cells was obvious increased. the negative control group compared with the liposome control group, P>0.05, statistically difference was not found.4 The Western-blot experiment: To test the E-cadherin and N-cadherin expression in each group of cells after transfection 48 h,the expression of E-cadherin: the experimental group(0.273 ± 0.045), the negative control group(0.645±0.020),the liposome control group(0.594±0.031) and the blank control group(0.390±0.034).The experimental group compared with the other 3 control groups,P<0.05,the difference was statistically significant, the expression of E-cadherin in the experimental group was decreased than other 3 groups. The expression of N-cadherin: experimental group(0.679±0.032), negative control group(0.578±0.030), liposome control group(0.576±0.013) and the blank control group(0.581±0.020). The experimental group compared with the other 3 control groups,P<0.05,the difference was statistically significant, the expression of N-cadherin in the experimental group was increased. But compare between the 3 control group, P>0.05,there was no significant statistically difference.Conclusions:1 Twist gene overexpression in human choriocarcinoma JEG-3 cells can make a change in the cell morphology, from round into irregular and the cell outline were unclear, presents the phenotypic characteristics of mesenchymal cells,that make choriocarcinoma JEG-3 cells has a more malignant invasion ability.2 Overexpression the Twist gene can increase the proliferation ability of human choriocarcinoma JEG-3 cells,so that accelerate its clinical developing process.3 The overexpression of Twist gene in human choriocarcinoma JEG-3 cells can increase the expression of N-cadherin and decrease the expression of E-cadherin. Illustrate that the Twist gene played an important role in the epithelial-mesenchymal transition of choriocarcinoma cells. |
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