| Background and ObjectiveSuccessful pregnancy depends on the orderly progress of fertilization,implantation,decidualization,placenta formation and delivery.As a specific organ between the mother and the fetus for blood exchanging,the placenta plays a key role in the maintenance of the normal pregnancy and the fetal development,therefore the placental formation is one of the most critical stages during the whole pregnancy.During the placenta formation stage,the differentiation of trophoblasts initially happens after successful implantation.The trophectoderm of embryo differentiate into three different kinds of trophoblast:cytotrophoblast(CT),syncytiotrophoblast(ST)and extravillous trophoblast(EVT),which respectively be in charge of differentiation,secretion and invasion.EVT is differentiated from CT and gradually invades into the decidua,even deep to decidual muscle layer.After invading into decidua,the EVT not only replaces the endothelial cells of the decidual spiral arteries so that helping remodel them into more receptive vessels which can sustain large amount of blood and high speed of the blood flow,but also directly interacts with the decidual cells to regulate the physiological characteristics of the decidual cells in order to improve their acceptance of embryo cells.When EVT interact with decidual immune cells through receptor-ligand response,the immune toxicity of decidual immune cells will be down regulated,thus ensuring the establishment of maternal immune tolerance.As a semi-allograft,only when the protection of maternal immune tolerance can the embryo survive and developed healthily.Therefore,in-depth study of the molecular interaction between EVT and decidual immune cells in early pregnancy,and understanding of the significance of the cross-talk between maternal-fetal interface in the establishment of immune tolerance in early pregnancy is of great importance in improving the survival rate of embryo and the success rate of pregnancy.c-Fos is a major member of the Fos sub-family of the AP-1 transcriptional factors family.It participates in the regulation of various cellular physiological processes such as cell differentiation,invasion,migration and apoptosis,and is key regulatory factor in many systems such as peripheral immune system,skeletal system,nervous system,etc.In the progress of pregnancy,c-Fos also plays an essential role in embryonic development and trophoblast invasion.On the one hand,c-Fos can regulate the invasion and migration ability of EVT by adjusting the expression of several metalloproteinases;on the other hand,c-Fos can directly affect embryonic development by influencing the brain and bone development,and even directly decide its survival.However,although there are many studies focusing on the relationship between c-Fos and trophoblast migration ability and embryonic development,the relationship between c-Fos and reproductive immunity still remains unclear.As an important immunomodulatory transcriptional factor,c-Fos is known to play an important role in peripheral immune system,and our previous bioinformatics analysis predicted that c-Fos may bind to the distal enhancer of HLA-G,an important immune regulator on EVT,therefore regulating the immune state at maternal-fetal interface by indirectly adjusting the expression of HLA-G.Focusing on the problems above,this study intends to explore the regulation of c-Fos on the immune regulatory ability of EVT and its possible mechanism,so as to provide a theoretical basis for a better understanding of the molecular interaction at the maternal-fetal interface and the establishment of maternal immune tolerance in early pregnancy,and to offer a knowledge basis for the prevention and targeted treatment of pregnancy-related diseases.Material and MethodsThe expression of c-Fos in normal fresh early pregnant villi was detected by tissue immunochemistry,and the expression of c-Fos in JEG-3 cells was detected by cellular immunofluorescence.c-Fos targeted siRNA was designed and constructed to inhibit the expression of c-Fos in JEG-3 cells.PCR and Western Blot were used to detect the inhibition efficiency of siRNA.Scratching assay,Transwell assay and MTT assay were used to identify the changes of viability and invasion and migration ability of JEG-3 cells after inhibition of c-Fos,as well as verifying whether the siRNA system can influence the biological function of JEG-3.Then,PCR and Western Blot were used to detect the changes of HLA-G expression of JEG-3 after successful c-Fos inhibition.Gene Ontology and gene enrichment analysis were conducted after high-throughput sequencing of whole transcriptome in order to detect the expression changes of genes in immune-related pathways after knockdown of c-Fos in JEG-3 Multi-factor analysis was used to detect the secretion level changes of Thl/Th2-related cytokines in JEG-3 cells after inhibition of c-Fos expression.Multi-color flow cell cytometry was used to detect the changes of peripheral blood cells(PBMCs)components after co-cultured with JEG-3 whose c-Fos expression was inhibited.Results(1)ENCODE prediction analysis showed that there are probably a c-Fos binding sites in the distal enhancer region of HLA-G.(2)Immunocytochemistry and cellular immunofluorescence of villi and JEG-3 showed that c-Fos was highly expressed in normal early pregnant villi and JEG-3 cell lines.(3)PCR and Western Blot after c-Fos targeted siRNA transfection into JEG-3 cells showed that co-transfection of three siRNA can better inhibit the expression of c-Fos(inhibition rate of mRNA expression:39%± 0.015,P<0.001,inhibition rate of protein expression:33.86%±0.034,P=0.030)than single transfection of any siRNA(mRNA expression inhibition:c-Fos-homo-685:12%±0.05,P=0.016;c-Fos-homo-816:8%± 0.04,P=0.035;c-Fos-homo-1002:25%±0.05,P=0.001);(4)Scratching,Transwell and MTT assay showed that inhibition of c-Fos could increase the invasion and migration ability(P=0.005)of JEG-3 cells,but had no significant effect on the viability of JEG-3(P>0.05).(5)High-throughput sequencing of whole transcriptome showed that c-Fos could influence the expression of genes in multiple immune pathways in JEG-3.(6)Multi-cytokine assay showed that inhibition of the expression of c-Fos in JEG-3 cells could significantly reduce its secretion of IL-6(P<0.001)and IL-4(P=0.015).(7)Direct co-culture of JEG-3 and PBMCs after the knockdown of c-Fos expression in JEG-3 showed that inhibiting c-Fos expression can downregulated the immune tolerance inducing ability of JEG-3;(8)PCR and Western Blot of HLA-G after c-Fos knockdown showed that inhibition of c-Fos expression might not affect the expression of HLA-G in JEG-3 cells(P>0.05).Conclusions(1)c-Fos can inhibit the invasion and migration of JEG-3 cells,but had no significant effect on the activity of JEG-3 cells;(2)c-Fos can promote the immune regulatory ability of JEG-3 probably by regulating the secretion of inflammatory cytokines of JEG-3 and expression of genes related to other immune pathway in JEG-3,rather than regulating the expression of HLA-G in JEG-3. |
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