Syncytin Contribute To The Pathogenesis Of Preeclampsia Through Regulating Trophoblast Cells

BackgroundAs we known,placenta is crucial for mammalian embryo development,and its integrity and normal functions depend on the precise regulation of multiple genes.Preeclampsia is a pregnancy-specific disease,and its pathogenesis has not been fully elucidated.Studies have shown that preeclampsia is a placental-derived disease,in which the placental pathological changes are manifested as trophoblast cell apoptosis,excessive autophagy,and reduced invasion,etc.These pathological changes would lead to placental‘superficial implantation’and placental disorder revascularization,which resulting in poor uterine-placental hypoperfusion and placental hypoxia.Syncytin is specifically expressed in placental trophoblast cells and mediates cell-cell fusion of cytotrophoblast cells into the syncytiotrophoblast layer.A decreased expression level of Syncytin was found in preeclampsia placenta,and the expression level of Syncytin was related to the severity of preeclampsia.In addition to its fusion function,Syncytin may also participate in the regulation of trophoblast cell biological function through its non-fusion functions in the occurrence of preeclampsia.This study intends to explore the possible mechanism of the effect of Syncytin on the biological function of trophoblast cells in the pathogenesis of preeclampsia.Material and methods1.Analysis of Syncytin expression in PE trophoblast cell model and sPE placentas.1.1 The mRNA and protein expression levels of Syncytin-1 in the control and PE trophoblast cells were detected by real-time quantitative PCR and western blotting respectively.1.2 The expression level of apoptotic proteins in the control and PE trophoblast cells were detected by western blotting.1.3 Histological analysis of Normal and sPE placentas.1.4 The expression of apoptotic proteins were detected in normal and sPE placentas by western blotting.2.Effects of conditionally induced syncytin-a gene knockout embryonic and placental development.Conditional knockout of syncytin-a gene mice were constructed and the knockout efficiency of syncytin-a gene was detected by real-time quantitative PCR.2.1 Phenotype of pregnancy mice:CCB assay was used to detect urinary protein of pregnant mice.2.2 Phenotypes of placentas and littermates:the phenotypes of placentas and littermates in the control and induction groups at different embryonic day（E14.5～E18.5）were recorded by stereoscopy.2.3 Morphological analysis of fetus:HE staining was used to observe fetal morphological changes in the Cre ER^T2/Syn A^fl/fland Cre ER^T2/Syn A^-/-groups.2.4 Placental transport capacity of the Cre ER^T2/Syn A^fl/fland Cre ER^T2/Syn A^-/-groups were detected by rhodamine 123 accumulation.2.5 Morphological analysis of placentas:HE staining,immunohistochemical staining,, immunofluorescence staining and transmission electronic microscopy were used to observe the morphological changes of placentas at different embryonic day（E14.5～E18.5 d）.2.6 Analysis of placental angiogenesis:immunofluorescence staining was used to observe the vessels,and the expression of PLGF,VEGF and s Flt-1 in the placenta were detected by real-time quantitative PCR.2.7 Analysis of placental apoptosis:TUNEL staining was used to detect placental apoptosis, and western blotting was used to detect the expression of apoptotic proteins in placentas.3.Effects of Syncytin on trophoblast biological functions.HERV-W siRNA was transfected into Be Wo and HTR-8/SVneo cells,and the HERV-W gene knockout cells were constructed.Real-time quantitative PCR and western blotting were used to detect the mRNA and protein expression levels of Syncytin-1.3.1 Tube formation was used to evaluate the effect of Syncytin-1 knockdown on angiogenesis in the trophoblast cells,and real-time quantitative PCR and ELISA were used to detect the expression changes of angiogenesis related factors,such as PLGF,VEGF and s Flt-1.3.2 MTT assay was used to detect the effects of Syncytin-1 on trophoblast cell proliferation, and real-time quantitative PCR was used to detect the mRNA expression of proliferation markers,such as PCNA and ki-67.3.3 TUNEL staining was used to detect the effect of Syncytin-1 on trophoblast cell apoptosis, and western blotting was used to detect the expression level of apoptotic proteins.3.4 Western blotting was used to detect the effect of Syncytin-1 on the expression of LC3B in trophoblast cells,and autophagy related genes Atg7,Atg4B and Beclin1 were detected by real-time quantitative PCR.3.5 Transwell migration and invasion assays were used to detect the effect of Syncytin-1 on the migration and invasion ability of trophoblast cells,wound healing assay was also used to detect the migration ability,and the mRNA expression levels of MMP2,MMP9 and TIMP were detected by real-time quantitative PCR.3.6 Expression changes of EMT related proteins E-cadherin and Vimentin were detected by western blotting.3.7 The effects of Syncytin-1 on the mRNA expression of TNF-αand IL-10 inflammatory cytokines in trophoblast cells were detected by real-time quantitative PCR.Results1.Expression of Syncytin-1 was significantly down-regulated in PE model of trophoblast cells compared with the control group.Compared with normal placentas,the morphology of sPE placentas were significantly changed,MVD was significantly decreased,and apoptotic cells were significantly increased.Also,the apoptotic proteins in PE trophoblast cells and sPE placentas were increased.2.Conditional knockout of syncytin-a gene in mice with placental dysplasia,placental vascular formation disorders,trophoblast apoptosis,abnormal maternal-fetal exchange, resulting in fetal intrauterine death during pregnancy.3.In vitro studies showed that down-regulated Syncytin-1 expression inhibited angiogenesis in vitro,and the expression of PLGF,VEGF were also decreased,while promoting the expression of s Flt-1 of trophoblast cells.At the same time,we found that the down-regulated expression of Syncytin-1 significantly inhibited the proliferation of trophoblast cells,and the expression of PCNA and Ki-67 were significantly decreased. Apoptosis of trophoblast cells increased and depended on Caspase pathway.The autophagy increased and the expression levels of Atg7,Atg4B and Beclin1 were increased.The migration and invasion abilities of trophoblast cells were decreased,and the expression levels of MMP2,MMP9 and TIMP were down-regulated.In addition,the expression level of Vimentin was significantly down-regulated while the expression of E-cadherin was unchanged in Syncytin-1-silenced trophoblast cells,suggesting that the down-regulated expression of Syncytin-1 might be involved in the EMT process.Down-regulated Syncytin-1 promoted TNF-αexpression and down-regulated IL-10 expression in trophoblast cells.ConclusionIn this study,the expression of Syncytin-1 was down-regulated and apoptosis was significant in PE trophoblast cell model.In vivo studies showed that syncytin-a gene knockout induced placental dysplasia,inhibited placental angiogenesis and resulted in the intrauterine death of fetal mice ultimately.Further in vitro studies confirmed that Syncytin-1 was involved in the process of PE placental dysplasia by regulating angiogenesis,proliferation,invasion,migration,EMT of trophoblast cells.