| The placenta is a chimeric organ that connects maternal and fetal structures and is essential for the maintenance and establishment of pregnancy.Placental trophoblasts differentiate into anchoring chorionic trophoblasts and extrachorionic trophoblasts that extend through the chimeric trophoblast layer and invade the maternal uterine tissue via the interstitial and intravascular pathways,remodeling the spiral arteries and establishing the uteroplacental circulation and subsequent oxygen and nutrient transport processes.However,inadequate EVT invasion into the uterine meconium and subsequent abnormalities in spiral artery remodeling can lead to adverse pregnancy outcomes such as spontaneous abortion and preeclampsia.The specific molecular mechanisms underlying the regulation of trophoblast differentiation are still unclear.In this study,human early pregnancy chorionic villus tissue,mouse placental tissue and human trophoblast cell lines were used in combination with relevant histomorphological analysis techniques,molecular biology and cell biology assays to investigate the process of trophoblast differentiation and the pathogenesis of spontaneous abortion,and the results of the study are as follows.Ⅰ.Physiological hypoxia is involved in the regulation of EVT differentiation via the downstream target genes of HIF1αFirstly,bioinformatics analysis of transcription factor HIF1αtarget genes and differentially expressed genes during EVT differentiation was performed,and the two intersecting genes were screened as potential target genes for transcription factor HIF1αto regulate EVT differentiation.The experimental validation was also performed using a physiological hypoxia model of trophoblast cells,RT-q PCR and Western blot.It was found that the expression of transcription factor HIF1αand its downstream target genes WIPF1,PLOD2,HPCAL1,SLC16A3,FAM174B and SYDE1 were up-regulated after physiological hypoxia（8%O2）treatment,while NREP and CD4 were down-regulated.After overexpression of HIF1α,the above genes were regulated and showed the same expression pattern as after physiological hypoxia treatment.Experimental validation of the expression of the above key factors using a trophoblast-induced differentiation model revealed that WIPF1 and PLOD2 were up-regulated during the differentiation of the trophoblast-invasive phenotype.It is suggested that physiological hypoxia regulates the downstream target genes of trophoblast through HIF1αto participate in EVT invasive differentiation process.Ⅱ.PLOD2 and WIPF1 promote the migration and invasion of trophoblast cellsGiven that trophoblast cells require the formation of actin cytoskeletal protruding structures pseudopod vesicles during successful invasion,while degrading the extracellular matrix complementarily.We further carried out subsequent studies from the extracellular matrix remodeling-related gene PLOD2 and the actin cytoskeleton remodeling-related gene WIPF1.First,based on the PLOD2 gene,we found that knocking down the targetgene PLOD2 significantly inhibited trophoblast invasion,migration and exosome outgrowth levels by Transwell invasion,cell scratching and villi exosome assays.To investigate the effect of PLOD2 on trophoblast EMT,we examined the expression levels of trophoblast EMT markers and found that interfering with PLOD2 inhibited trophoblast epithelial-mesenchymal transition.Secondly,for the WIPF1 gene,we likewise found that interference with WIPF1 produced inhibition of trophoblast invasion,migration and exosome outgrowth ability by the above experiments,however,overexpression of WIPF1 produced the opposite effect.To further investigate the molecular mechanism of WIPF1 involvement in trophoblast invasion and differentiation,we used overexpression of WIPF1 plasmid to transfect HTR-8/SVneo cells,and found WIPF1-interacting proteins by IP-MS.Using GO and KEGG analysis,the results showed that the WIPF1interacting protein was mainly associated with ACTN4,a key gene of the actin cytoskeleton,and Vimentin,an EMT-related gene.Cytoskeleton staining revealed that knockdown of WIPF1 interfered with cytoskeletal remodeling and formation of atypical pseudopod vesicles in trophoblast cells,while overexpression of WIPF1 produced the opposite effect.It is suggested that in PLOD2 and WIPF1 are involved in EVT migration,invasion and EMT processes and are important factors in regulating EVT function.Ⅲ.WIPF1 promotes trophoblast invasion via YAPWIPF1 RNAi and overexpression models of trophoblast cell lines HTR-8/SVneo and JEG-3 and Western blot method were used to detect the related EMT protein expression,and the results showed that WIPF1overexpression could promote trophoblast epithelial-mesenchymal transition（EMT）.Further,HTR-8/SVneo cells were induced by the EMT activator TGFβ1,and Western blot assay of protein levels revealed that WIPF1 and N-Cadherin were induced by TGFβ1,and the downregulation of WIPF1 levels due to knockdown of WIPF1 and its induced downregulation of N-Cadherin were rescued to promote trophoblast EMT.In addition,the trophoblast localization and protein expression levels of YAP andβ-catenin were examined by immunofluorescence and Western blot,and the results showed that nuclear expression and protein levels of YAP andβ-catenin were upregulated in control cells by overexpression of WIPF1,and conversely,nuclear expression and protein levels of YAP were downregulated in control cells by knockdown of WIPF1.Not only that,by treating trophoblast cells with YAP inhibitor VP,it was found that YAP inhibitor downregulated YAP and N-Cadherin in control group while upregulating E-Cadherin,and WIPF1 overexpression group could upregulate epithelial marker protein E-Cadherin in YAP inhibitor group and promote EMT in trophoblast cells.interestingly,HTR-8/SVneo cells knocking down and overexpressing WIPF1 and treated with TGFβ1revealed that TGFβ1-induced upregulation of YAP nuclear expression levels in the negative control plasmid group cells,knockdown WIPF1group cells.In addition,VP treatment of HTR-8/SVneo cells significantly reduced the actin backbone remodeling and the fluorescence intensity and number of atypical pseudopod vesicles in cells transfected with the negative control plasmid NC-m Cherry group and in cells overexpressing WIPF1 group.It is suggested that WIPF1 acts as an upstream factor to mediate the involvement of YAP in the EVT actin skeleton remodeling and EMT regulation process,and promotes trophoblast invasion.Ⅳ.Abnormal expression of WIPF1 is involved in the occurrence of spontaneous abortionUsing immunohistochemistry and immunofluorescence,WIPF1 was found to be highly expressed in early pregnancy villi but lowly expressed in aborted villi.Similarly,it was highly expressed at the location of trophoblast giant cell（TGC）labeled by PL1 at the maternal-fetal interface on the eighth day of gestation in normal mice,but lowly expressed in TGC of aborted mice.This suggests that abnormal expression of WIPF1 may be involved in the occurrence of spontaneous abortion.The present study confirmed that physiological hypoxia could induce the upregulation of HIF1αand further mediated the upregulated expression of its downstream target genes PLOD2 and WIPF1 to promote EVT differentiation,and its abnormal expression might be involved in the occurrence of spontaneous abortion,which provides a scientific basis to reveal the potential target factors and their molecular mechanisms in the pathogenesis of spontaneous abortion. |
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