The Effection Of The Invasion Of JEG-3 Cells Transfected By PPARγ SiRNA

[Background and Objective]Peroxisome proliferator activated receptors-gamma(PPARγ)is member of theⅡtype nuclear receptors superfamily, which the functions are complex, concerning with the metabolize of glucose and fat, the activation of mononuclear cells, inflammatory, the proliferation, differetiation and apoptosisof cells. As a result, PPARγis deeply in contact with physiologic and/or pathologic pregnancy. PPARγis necessary in the development of placenta,which is concern with the invasion,proliferation and migration of trophoblast. Recengtly, researches have revealed pathologic pregnancy such as abortion, preeclampsia and fetal growth restriction is related to biologic function of trophoblast. In our researches, we observed the suppression effects of PPARγby small interference RNA (siRNA), investigated the infection of the invasion of JEG-3 cell line, investigated the possible role of PPARγgene in the regulation of the mechanism of the invasion of trophoblast,and based on these,wo can go a step further to apprehend the pathologic pregnancy such as preeclampsia, fetal growth restriction and recurrent abortion.【Method】1. Culture JEG-3 cell line routinely.2. Based on the Genebank cDNA accession number, design and synthesize 3 PPARγsiRNA oligonucleotides lines. Design and synthesize 1 negative siRNA oligonucleotides,1 labelled with fluorescence negative siRNA oligonucleotides, which are not specific for any known human gene.3. Culture JEG-3 cell line routinely.Transfected the labelled with fluorescence negtive siRNA by the concentration of 100nmol/L,50nmol/L independently. Observe by fluorescence microscope to choose the more appropriate conceration.4. Culture JEG-3 cell line routinely.Transfect the siRNA to JEG-3 cells,5 groups was designed, negtive control, S1, S2, S3 and S4 (mixed) group.5. Real-time quantitative PCR (qRT-PCR) was used to detect the depletion of PPARγ6. Culture JEG-3 cell line routinely.Transfect the siRNA to JEG-3 cells,3 groups was designed, negtive, test and control group.7. Real-time quantitative PCR(qRT-PCR) was used to detect the expression of PPARγand MUC1 mRNA, respectively.8. Trans well invasive test: Transwell chambers were coated with Matrigel. Tranfected after 24h, cells was harvested with RPMI-1640 media which was supplemented with 10% FCS in the upper chambers respectively. RPMI-1640 media supplemented with 20% FCS was put in the lower chambers. After incubated for 24 hours, the cells which had penetrated through to the bottom of the chamber were counted by microscope (40xfield).9. Statistical analysis: The quantitative datas of our experiments were figured as mean±standar deviation(X±s), and analysed by one-way analysis of variance test, using SPSS 17.0 for Windows.【Results】1. Observed by fluorescence microscope, the efficiency of transfection is higher in the concertration of 100nmol/L than in the concertration of 50nmol/L.2. Compered with the control group, PPAR Y siRNA transfection resulted in (40.88±1.05)% decrease of PPAR Y mRNA level in group S1, a (71.92±0.71)% decrease of PPARγmRNA level in group S2, a (62.69±0.66)% decrease of PPAR Y mRNA level in group S3, a (52.8±0.52)% decrease of PPARγmRNA level in group S4 (p<0.05)3. Compered with the control group, PPAR Y siRNA transfection resulted in a (75±0.8)% decrease of PPARγmRNA level (p<0.05),and a (65±1.3)% decrease of MUC1mRNA level (p<0.05).4.JEG-3 cellS invasion capacity was significantly strengthened, compared with the other two groups (p<0.05).[Conclusion]PPARγsiRNA can inhibit the expression of PPARγin JEG-3 cells,and decrease the level of MUC1, strengthen the invasion of JEG-3 cells.Then we may presume that PPARγmay regulate the invasion of trophoblast by MUC1,and based on these we can go a step further to understand the mechanism of the pathologic pregnancy such as preeclampsia, fetal growth restriction and recurrent abortion.