The Effection of the Invasion of JEG-3 Cells Transfected by PPARγ siRNA
|Course||Obstetrics and Gynaecology|
|Keywords||Small RNA interference Peroxisome proliferator-activated receptor γ Mucin MUC1 JEG-3 Trophoblast invasion force|
[Background and Purpose] peroxisome proliferator-activated receptor γ (peroxisome proliferator activated receptors-gamma, PPARγ) is a type II nuclear receptor superfamily members, and its function is more complex, involved in the metabolism of sugar and fat, single nuclear cell activation, inflammation, cell differentiation, proliferation and apoptosis and other physiological and pathological processes. PPARγ in placental development is required to participate in the process of trophoblast invasion, proliferation, migration, and therefore is closely linked with the physiological and pathological pregnancy. In recent years, many studies have shown that preeclampsia, fetal growth restriction and pathology of recurrent spontaneous abortion pregnancy occurred with trophoblastic invasion ability abnormalities. The purpose of this study is to use small molecule RNA interference in JEG-3 in the expression of PPARγ in human choriocarcinoma cell line to study its impact on in vitro trophoblast invasion force of their participation in the regulation of trophoblast invasion mechanism for eclampsia the early stage of fetal growth restriction and recurrent spontaneous abortion pathological pregnancy pathogenesis provide a theoretical basis.  1. Conventional recovery, cultured human choriocarcinoma cell line JEG-3 to spare logarithmic phase. 2 according to the sequence number of the Genebank of PPARγ, designed and chemically synthesized PPARγsiRNA for to three specific PPARγsiRNA, S1, S2,, S3,; non-specific negative control siRNA, 1 fluorescently labeled non-specific negative control siRNA. Conventional culture JEG-3 cells, planted in a 6-well plate, the fluorescently labeled blank control siRNA to a final concentration 100nmol / L respectively, 50nmol / L of transfected cells, the transfection efficiency was observed with a fluorescence electronic microscope, selection The optimum concentration of siRNA transfection. Routinely cultured JEG-3 cells, planted in six-well plates, experimental ① divided into negative control group (transfected with negative control siRNA), S1 group (PPARγ-specific siRNA transfection the S1 chain of), S2 ( Transfected S2 in PPARY specific siRNA chain), S3 group (PPARγ specific siRNA transfection S3 chain), S1, S2, S3 S4 group (transfected with the mix of PPARγ-specific siRNA strand) carried transfection. Using real-time quantitative PCR the technique detection the level of PPARγmRNA after transfection of the cells in each group, and select the highest group of PPARγ inhibition efficiency. 6. JEG-3 cells routinely cultured to logarithmic phase species in six-well plates, and experimental ② divided into an experimental group (transfected PPARγ specificity siRNAS2 chain), negative control group (transfected with negative control siRNA), blank control group (not transfected with siRNA, I reagents with the other two groups of the same), and transfection. Using real-time quantitative PCR to detect the level of the three groups of cells transfected PPARγmRNA to detect the rate of siRNA inhibition of PPARγ and mucin MUC1 mRNA level was measured. The 8. Transwell cell invasion assay: three groups of transfected cells after 24 hours, with 10% fetal calf serum (fetal cow serum, FCS), resuspended culture liquid, seeded in the upper chamber of the package is small chamber matrigel Matrigel, The lower chamber was added 20? the S culture liquid, after 24 hours incubation, the conventional HE staining, under a microscope (40 times) taken from five fields of view count the number of penetrating cells for detecting invasiveness of JEG-3 cells after transfection PPARγsiRNA. SPSS 17.0 statistical methods: application software package for statistical analysis and expressed as mean ± standard deviation (X ± s) quantitative data about data, groups were compared using one-way ANOVA, to think P lt; 0.05 was statistically significant. [Results] 1. Fluorescence under an electron microscope observation to a final concentration of 100nmol / L fluorescently labeled siRNA transfection efficiency. 24 hours after transfection, compared with the negative control group, the S1 group PPARγmRNA level down (40.88 ± 1.05)% (p lt; 0.05) (71.92 ± 0.71)%; the S2 group PPARγmRNA level down; S3 group PPARγmRNA downregulated (62.69 ± 0.66)%; the S4 group PPARγmRNA level down (52.8 ± 0.52)%. Come the S2 group PPARγsiRNA inhibition most efficient. 24 hours after transfection, compared with the blank control group, the experimental group PPARγmRNA expression level reduced by (75 ± 0.8)% (p lt; 0.05), the expression of the negative control group PPARγmRNA no difference (p gt; 0.05) . MUC1 mRNA expression levels in the cells in each group, the experimental group compared with the blank control group lowered (65 ± 1.3)% (p lt; 0.05), negative control group and blank control group, there was no statistically significant difference (p gt; 0.05) . The number of cells. Compared with the blank control group, the experimental group After transfection PPARγsiRNA, in JEG-3 cells to penetrate Metrigel Matrigel significantly increased, and the difference was statistically significant (p lt; 0.05), negative control group and blank control group comparison there was no statistically significant difference. [Conclusion] siRNA technology can effectively inhibit cancer cell lines of human chorionic expression in JEG-3 cells PPARγmRNA downward MUC1 mRNA expression, and can enhance the ability of trophoblastic invasion, suggesting that PPARγ gene may regulate MUC1 gene impact of trophoblast invasion, which involved in the occurrence of pathological pregnancy such as preeclampsia.