| Placenta is an important place for material exchange between fetus and mother,and trophoblast is the main cell type,which plays a crucial role in embryo implantation and placental development.Cytotrophoblast invades the spiral artery of the uterus and differentiates into villous trophoblast cells,causing the remodeling of uterine and placental blood vessels.At 10 to 12 weeks of gestation,extravillous trophoblasts form plugs that occlude the spiral arteries and prevent maternal blood from entering the intervillous space,which creates a low oxygen in placenta（less than20mmHg）,then,plugs disintegrate and maternal blood gets into the placenta cavity circulation,which oxygen pressure rises to 5060mm Hg.During this period,oxygen concentration plays a key role in the invasion of trophoblastic cells.The research indicates that insufficient trophoblast invasion cells causes the failure of spiral artery remodeling,which leads to many kinds of pregnancy diseases,such as abortion,stillbirth,preeclampsia,fetal intrauterine restriction,etc.Hypoxia inducible factor-1α（HIF-1α）is a transcription factor that widely expressed in human and mammal under hypoxic condition.It can regulate transcription of many downstream target genes,and plays an important role in maintaining and regulating gestational trophoblastic behavior after its activation.Meanwhile,there are considerable similarities,in the process of normal pregnancy,between the invasion and migration of trophoblast cells and the infiltration and distant metastasis of tumor cells to surrounding tissues,but the invasion of trophoblastic cells is strictly limited by time and space.At the same time,the interaction of trophoblast and maternal oxygen level during the development of the placenta is an important factor for regulating the invasion process.Chemokine receptor 4（CXCR4）is a G protein coupled receptor composed of352 amino acids.After binding to its ligand CXCL12,it can activate downstream multiple signal transduction pathways to regulate the invasion and migration of gestational trophoblastic cells.Studies have shown that HIF-1 alpha can directly bind to the hypoxia responsive elements of the CXCR4 gene promoter in osteosarcoma cells under hypoxic conditions,upregulating the expression of CXCR4 and promoting the invasion and metastasis of tumor cells.However,the relationship of HIF-1α,CXCR4 and the invasion and migration of trophoblast in hypoxic has not been reported.ObjectiveReal-time quantitative PCR,Western blot,Transwell test and Wound scratch assays were used to detect the mRNA and protein expression of HIF-1αand CXCR4in human chorionic trophoblast cell line JEG-3 cells under hypoxic conditions,and the effect on trophoblast invasion and migration ability.The effect of HIF-1αon the expression of CXCR4 in trophoblast cells and the invasion and migration of cell were investigated.Materials and Methods1 MaterialsHuman chorionic trophoblast cell line JEG-3 cells purchased from the Beijing DingGuo Changsheng Biotechnology were cultured with RPMI-1640 complete medium（RPMI1640,10%FBS,100U/ml penicillin,100U/ml streptomycin）under the moist condition of 37℃,5%CO2.JEG-3 cells in exponential growth stage were used for all exposure experiments.2 MethodsRPMI1640 complete medium was used to suspend JEG-3 cell cultured in constant temperature incubator,when cells reached the exponential phase,they were seeded into bottles or six-well plates,and then continued to culture.When cells reach70%,they were used to do subsequent experiments.2.1 The effect of hypoxia on the expression of HIF-1αand CXCR4 in JEG-3 cells150μmol/L Cobalt chloride（CoCl2）was used to establish a hypoxia model of12h,24h and 48h of JEG-3 cells in vitro,respectively.The cells were divided into the normal oxygen control group,hypoxia 12h group,hypoxia 24h group and hypoxia48h group.Real-time PCR and Western blotting were used to measure the m RNA and protein expression levels of HIF-1αand CXCR4 in JEG-3 cells,respectively.2.2 The effect of HIF-1αsilencing on the expression of CXCR4 and the invasion and migration of JEG-3 cells in hypoxiaJEG-3 cells were divided into 4 groups:normoxia control group（without CoCl2）,hypoxia control group（CoCl2 treatment）,hypoxia NC group（transfected with HIF-1αnegative control then CoCl2 treatment）,hypoxia HIF-1αsiRNA group（HIF-1αsiRNA transfection then CoCl2 treatment）.Real-time PCR and Western Blot were used to detect the mRNA and protein of HIF-1αand CXCR4,and Transwell assay and Wound scratch assay were used to detect the cell invasion and migration.3 Statistics analysisSPSS 21.0 were adopted to analyze the experimental data.Experimental data were expressed as mean±SD,and differences among different groups were compared using ANOVA.LSD-t test was used to further compare the difference between any two groups.α=0.05 was considered statistically significant.Results1Expressions of HIF-1αand CXCR4 mRNA in JEG-3 cells in hypoxiaReal-time PCR showed that the relative expression of HIF-1αmRNA in hypoxia12h group（1.16±0.25）,hypoxia 24h group（1.07±0.12）and hypoxia 48h group（1.05±0.17）had no significant change compared with normoxia control group(1.03±0.15,and the difference was not statistically significant（P>0.05）.The relative expression level of CXCR4 mRNA increased with the prolongation of hypoxia time.Hypoxia12h group（4.84±0.28）,hypoxia 24h group（7.12±0.65）and hypoxia 48h group（11.19±0.91）were significantly higher than those in normoxia control group（1.05±0.16）,the differences were statistically significant（P<0.05）.2Expression level of HIF-1αand CXCR4 protein in JEG-3 cells in hypoxiaWestern Blot showed that the extension of the expression level of HIF-1αand CXCR4 protein were increased with the time of hypoxia:the protein expressions of hypoxia 12h group（0.66±0.06,0.60±0.04）,hypoxia 24h group（0.91±0.04,0.84±0.07）,hypoxia 48h group（1.11±0.09,1.04±0.07）higher than those of normal oxygen control group（0.26±0.07,0.38±0.16）,andthedifferenceswerestatistically significant（P<0.05）.3Expression of HIF-1αand CXCR4 mRNA in JEG-3 cells after HIF-1αsilencing under hypoxic conditions3.1 Expression of HIF-1αmRNA in JEG-3 cells after silencing HIF-1αunder hypoxic conditionReal-time PCR showed that:HIF-1 mRNA expression level（0.23±0.08）in hypoxia HIF-1αsiRNA group was significantly lower than hypoxia blank group（0.98±0.06）,hypoxiaNCgroup（1.03±0.09）,andnormoxiablank group（1.00±0.11）,which the difference was statistically significant（P<0.05）.3.2 Expression of CXCR4 mRNA in JEG-3 cells after silencing HIF-1αunder hypoxic conditionThe expression level of CXCR4 mRNA in hypoxia HIF-1αsiRNA group（0.44±0.08）was significantly lower than that in hypoxia blank control group（6.18±0.79）,hypoxiaNCgroup（5.83±0.33）andnormoxiablank group（1.35±0.10）,which the difference was statistically significant（P<0.05）.The expression of CXCR4 mRNA in the hypoxia blank group and hypoxia NC group was higher than that in the normoxia blank group,and the difference was statistically significant（P<0.05）.4Expressions of HIF-1αand CXCR4 protein in JEG-3 cells after silencing HIF-1αunder hypoxic conditionWestern Blot showed that the protein expression levels of HIF-1αand CXCR4in hypoxia HIF-1αsiRNA group（0.13±0.05,0.30±0.03）were significantly lower than those of hypoxia blank group（0.88±0.09,0.96±0.06）,hypoxia NC group（0.82±0.11,0.87±0.07）and normoxia blank group（0.29±0.08,0.44±0.07）,and the differences were statistically significant（P<0.05）.The expression levels of HIF-1αand CXCR4 protein in hypoxia blank group and hypoxia NC group was higher than those of normoxia blank group,and the difference was statistically significant（P<0.05）.5Changes of the invasion and migration in JEG-3 cells after silencing HIF-1αunder hypoxic conditionThe numbers of invasive cell and scratch width healing rate in hypoxia HIF-1αsiRNA group（34.93±8.01,25.08±4.73）were significantly lower than those in hypoxiablankgroup（88.40±5.81,74.63±4.90）,hypoxiaNCgroup（82.67±4.28,70.22±3.05）and normoxia blank group（46.53±4.70,43.89±4.05）,and the differences were statistically significant（P<0.05）.The numbers of invasive cell and scratch width healing rate in hypoxia blank group and hypoxia NC group were higher than those in normoxia blank group,and the difference was statistically significant（P<0.05）.ConclusionsHypoxia promotes the expression of HIF-1αand CXCR4,and silencing of HIF-1αmay inhibit the invasion and migration of trophoblast through downregulation of CXCR4. |
