Sulg Gene Induced Epithelial-mesenchymal Transition Of Choriocarcinomal Cells

Objective:Choriocarcinoma,short for CC,Is a highly malignant gestational trophoblastic disease,which Can be secondary to hydatidiform mole pregnancy,abortion,full-term pregnancy,ectopic pregnancy.Its characteristic is the trophoblast cells lost the original structure,Invading muscularis.It has highly aggressive,rapid growth and can metastasize to the vital organs such as lung and brain in early stage.Due to its extremely sensitive to chemotherapy drugs,the majority of patients can be treated by chemotherapy.But the drug resistance seriously affect the process of conquering choriocarcinoma.epithelial-mesenchymal transition?EMT?,referring to the change of morphological characteristics in the epithelial cell in the certain parts,makes it be suitable for invasion and metastasis in biology.In recent years a large number of experiments show that EMT participates in the occurrence and development of various epithelial malignant tumor.Slug as one of zinc finger transcription factor family,is an important regulatory factor of EMT.Through combining with E-box of E-cadherin it can inhibit its intracellular transcription,destroy the close connections between cells,thus inducing the occurrence of EMT.This experiment studys E-cadherin and Ncadherin expression level after overexpression of Slug genes in choriocarcinoma JEG-3 cells,states the occurrence and development mechanism of choriocarcinoma and provides theoretical basis for the treatment.Method:1 Human choriocarcinoma JEG-3 cells,at 37 ?,5% CO₂ thermostatic incubator to cultivate.2 Experimental groups:?1?the experimental group,transfected p EGFP-N1-Slug plasmid;?2?the negative control group,transfected blank plasmid;?3?liposome control group,transfected Lipofectamine^TM2000 liposome transfected reagent;?4?blank control group,regular training,without processing.3 Observe the cell morphological changes after transfection by Microscope.4 Detect the proliferation of cell at 0 zero hour,24 hours,48 hours,72 hours,and 96 hours by CCK 8 method.5 Detect the Slug-mRNA expression level of experimental group and the control group after 48 hours by Realtime-Qpcr.6 Test E-cadherin and N-cadherin expression level of cells after transfection 48 hours by Western blot.7 Data statistics analysis: using SPSS 13.0 statistical software for statistical analysis and experimental data was recorded ?±s.Means comparison between groups using single factor analysis of variance?one-way ANOVA?,differences between groups using LSD/SNK method.Inspection level ? =0.05,P < 0.05 means the difference was statistically significant,P > 0.05,means no statistically significant difference.Results:1 Observation of JEG-3 cell morphology:Observing cell morphology of each group,the cells of blank control group,liposome control group,negative control group are circular or elliptic,that have integral cell membrane;the cell of experimental group is irregular,fusiform or polygon,that arranged loosely.2 Observation of transfection efficiency:Useing of Lipofectamine^TM2000 liposome transfect JEG-3 cells,and observing transfection efficiency in luorescence microscope.The transfection efficiency of experimental group and the negative control group after transfection 24 and 48 hours are 15-20%,and 70-80%.Liposomes control group and blank control group without fluorescence expression.3 CCK 8 method to detect cell proliferation:0 hour : experimental group is 0.247±0.004,control group is 0.138±0.003,liposome control group is 0.143±0.003,Blank control group is 0.139±0.002,Comparing between groups?P > 0.05?there was no statistically significant difference.24 hour : experimental group is 0.247±0.004,control group is 0.244±0.007,liposome control group is 0.193±0.004,Blank control group is 0.221±0.010,The experimental group compared with control group,P > 0.05,there was no statistically significant difference,the experimental group compared with blank control group,liposomes,control group,P < 0.05,the difference was statistically significant.48 hour : experimental group is 0.509±0.004,control group is 0.417±0.003,liposome control group is 0.407±0.011,Blank control group is 0.418±0.009,The experimental group compared with the other three groups?P < 0.05?the difference was statistically significant.The multiplication rate of experimental group is faster than the other three groups.72 hour : experimental group is 0.977±0.019,control group is 0.617±0.003,liposome control group is0.515±0.013,Blank control group is 0.781±0.016,The experimental group compared with the other three groups?P < 0.05?the difference was statistically significant.The multiplication rate of experimental group is faster than the other three groups.96 hour : experimental group is 1.371±0.023,control group is 1.163±0.006,liposome control group is 0.801±0.005,Blank control group is 1.293±0.003,The experimental group compared with the other three groups?P < 0.05?the difference was statistically significant.The multiplication rate of experimental group is faster than the other three groups.4 Test the expression level of Slug-mRNA using Realtime-qPCR :experimental group is 2.070±0.028,control group is 1.093±0.011,liposome control group is 1.055±0.025,Blank control group is 1.002±0.021,The Slug-mRNA expression level of experimental group compared with the other groups is significantly rised?P < 0.05?the difference was statistically significant.5 Test the expression level of E-cadherin and N-cadherin in each groups by western blot after 48 hour of transfection:the expression level of E-cadherin: experimental group is 0.302±0.016,control group is 0.645±0.020,liposome control group is 0.594±0.031,Blank control group is 0.390±0.034,The E-cadherin expression level of experimental group compared with the other groups is significantly lowered?P < 0.05?the difference was statistically significant.the expression level of N-cadherin: experimental group is 0.672±0.037,control group is 0.578±0.030,liposome control group is 0.576±0.013,Blank control group is 0.581±0.020,The N-cadherin expression level of experimental group compared with the other groups is significantly up-regulation?P < 0.05?,the difference was statistically significant.Conclusion:1 The high expression of Slug genes can change the morphology of JEG-3,that make it more fit for the mesenchymal cell,making it a vicious attack force.2 High expression of Slug gene can improve proliferation rate of JEG-3,so as to accelerate the clinical process.3 The high expression of Slug genes can make expression level of E-cadherin decreased,expression level of N-cadherin rise,That means Slug genes are involved in EMT,which affect the occurrence and development of cancer.