Snail Gene Induced Epithelial-mesenchymal Transition Of Choriocarcinoma Cells

Objective: Choriocarcinoma(CC),short for CC, is a highly malignant tumor that grows rapidly. The main characters of choriocarcinoma are to spread through blood, to metastasis early and widely and to be sensitive to the chemotherapy. But the etiology is still not clear. The metastasis and chemoresistance of choriocarcinoma are key points which lead to the death of patients. Therefore, how to inhibit the invasion and migration of tumor cells is the researching hot topic.Epithelial-mesenchymal transition(EMT) refers to the process that epithelial phenotype cells having polarity rearrange the cytoskeleton, increase abilities of migration and the invasion, change the expressions of the epithelial cell marker E-cadherin and the mesenchymal cell marker N-cadherin, and transform into mesenchymal phenotype cells which possess the movement ability in particular physiological and pathological circumstances. The emerging date suggests that this important process is initially recognized during the embryonic development and orgnogenesis. Furthermore, it plays an important role in the local invasion and distant metastasis in a variety of epithelial tumors. Therefore, studying further of EMT molecular modulation and tumorigenesis, and then interferencing EMT by some technologies, may be a promising approach for the experimental of choriocarcinoma.Snail gene is first described in drosophila melanogaster. Subsequently, Snail and Snail homologs have been found in a lot of vertebrates including humans. Snail and Snail homologs belong to SNAIL family, including Snail, Slug, S1P1 and so on. The study about kinds of tumors has clearly demonstrated that Snail gene can induce EMT which is associated with the repression of E-cadherin and the induction of N-cadherin expression and increase abilities of migration and the invasion of tumor cells. However, little news is reported about the relationship of Snail gene and EMT of choriocarcinoma.In this study, with the choriocarcinoma JEG-3 cells cultivated in vitro, Snail gene transfection as the factor,we observed the differences of the cell morphology, migration capabilities, expressions of Snail-m RNA and EMT molecular markers, and aimed at studying the function of Snail gene in the occurrence and development in choriocarcinoma, furthermore, we explored the molecular mechanisms, intending to provide new ideas and theoretical basises for the experimental of choriocarcinoma.Methods:1 Cultivate the human choriocarcinoma cell line of JEG-3;2 The group of experiments:①though the Lipofectamine?2000 liposome transfection reagent,the experimental group was transfected with p EGFP-N1-Snail plasmid;②the negative control group was transfected with the empty plasmid;③the liposome control group was transfected with Lipofectamine?2000 liposome transfection reagent;④the blank control group that was the group without any treatment;3 The morphological changes of JEG-3 cells were observed under inverted microscope;4 The migration capabilities of JEG-3 cells in different groups were evaluated by using a wound healing assay;5 Real-time q PCR: the Snail-m RNA expression levels of JEG-3 cells in different groups were examined;6 Western blotting: expression changes of the epithelial cell marker E-cadherin and the mesenchymal cell marker N-cadherin were examined;7 All figures were statistically analysed by SPSS 13.0 statistical software. Data presented as mean±standard differential(`c±s),means of multiple groups were analyzed by using the single factor analysis of variance(one-way ANOVA),P>0.05 for no significant difference,P<0.05 for a significant difference. All the experimental results were repeated three times.Results:1 Results of the inverted microscope observationJEG-3 cells of three control groups were oval or round, connected tightly and gathered into a sheet growth which showed the morphology of epithelial cells;JEG-3 cells of the experimental group showed the fusiform cell shape, lost the cell polarity and arranged indisorder, which performanced characteristics of mesenchymal cells.It proved that Snail gene caused the mesenchymal transition of JEG-3 cells and increased the ability of the malignant invasion of JEG-3 cells.2 The transfection efficiencies were observed:After using Lipofectamine?2000 liposome transfection method transfection,the transfection efficiency was observed by the laser confocal microscope. After 24 hours both the experimental group and the negative control group had a weak fluorescent expression(transfection rate 15%~20%),and the fluorescent expression was enhanced after 48h(transfection rate 70%~80%).3 Results of cells scratch experimentThe cells continued to cultivate for 24 h after the scratch, with the microscope, we observed that the scratch healing area of the experimental group(47.3±2.32)% was more than that of the blank control group(14.9±1.21)%, P<0.05,while the difference was statistically significant. The above prompted that Snail gene enhanced the migration ability of JEG-3 cells;After the scratch 12 hours, the growth rate of the experimental group was(13.04±0.72)%,and the growth rate of the blank control group was(9.78±0.35)%, P>0.05, while the difference was not statistically significant. The growth speeds of the experimental group and the blank control group were similar;After the scratch 24 hours, the growth rate of the experimental group was(19.13±1.07)%,and the growth rate of the blank control group was(17.59±1.02)%, P>0.05, while the difference was not statistically significant. The growth speeds of the experimental group and the blank control group were similar.4 After transfection 48 hours, the expression levels of Snail-m RNA of JEG-3 cells in groups were detected by Real-time q PCRThe expression levels of Snail-m RNA in groups were detected: The experimental group was(2.067±0.031),the negative control group was(1.093±0.011),the liposome control group was(1.055±0.025),the blank control group was(1.002±0.021).The Snail-m RNA expression of the experimental group was increased than the negative control group, the liposome control group and the blank control group,P<0.05.The difference was statistically significant. The negative control group was compared with the liposome control group, P>0.05, there was no statistically difference.5 After transfection 48 hours,the expressions of E-cadherin and N-cadherin of JEG-3 cells in groups were tested by the Western blottingThe expressions of E-cadherin of JEG-3 cells in groups were tested: The experimental group was(0.268±0.034),the negative control group was(0.645±0.020),the liposome control group was(0.594±0.031) and the blank control group was(0.390±0.034).The expression of E-cadherin in the experimental group was decreased than other 3 control groups,P<0.05.The difference was statistically significant. Any two groups were compared with each other in the three control groups, P>0.05.There was no significant statistically difference.The expressions of N-cadherin of JEG-3 cells in groups were tested:The experimental group was(0.664±0.009),the negative control group was(0.578±0.030),the liposome control group(0.576±0.013) and the blank control group(0.581±0.020). The expression of N-cadherin in the experimental group was increased than other 3 control groups,P<0.05.The difference was statistically significant. Any two groups were compared with each other in the three control groups, P>0.05.There was no significant statistically difference.Conclusions:1 Snail gene changed the cell morphology of choriocarcinoma JEG-3 cells which presented phenotypic characteristics of mesenchymal cells, and increased the ability of the malignant invasion of JEG-3 cells.2 Snail gene enhanced the migration ability of choriocarcinoma JEG-3 cells, and promoted the metastasis of choriocarcinoma.3 Snail gene decreased the expression of E-cadherin and increased the expression of N-cadherin, which proved that the Snail gene played a role in the occurrence and development of choriocarcinoma through EMT.