WNT2 Expression And Its Epigenetic Regulation In Trophoblast Dysfunction Of Patients With Preeclampsia

BackgroundPreeclampsia(PE)is a specific disease in pregnancy,affecting up to 2%～8%of pregnant women worldwide.PE is characterized by hypertension and proteinuria after 20 weeks of gestation.Preeclampsia is a major cause of increased morbidity and mortality of pregnant and perinatal women and infants.Therefore,there is a need to identify the early diagnosis of preeclampsia and to develop efficient treatment strategies.The pathogenesis of preeclampsia is quite complex,which has not been fully elucidated yet.At present,there are many theories about the pathogenesis of pre-eclampsia,among which“the theory of poor recasting of spiral uterine arterioles and superficial placental implantation" is the most recognized by the academic circle.According to this theory,in the early stage of placenta formation,abnormal proliferation and differentiation of trophoblast cells,accompanied by excessive apoptosis,result in decreased infiltration and limited invasion into the uterine wall.These changes in trophoblast cells lead to insufficient uterine spiral artery recasting and shallow placental implantation,leading to the onset of preeclampsia.The successful maintenance of pregnancy depends on the participation of multiple genes and signal transduction pathways.Many studies have found that in the fonmation and development of placenta Wnt signaling pathway plays an important role.Wnt2 is an important member of the Wnt gene family and is the second Wnt gene to be identified.In animal studies,targeted destruction of the Wnt2 gene in mice resulted in decreased birth weight in their offspring,and half of them died in perinatal period.In addition,mutations in the Wnt2 gene lead to decreased capillaries and increased fibrous deposition in the fetus,and to placental abnormalities associated with placental angiogenesis defects.In placental formation,the ability of trophoblast cells to proliferate,differentiate and migrate and to avoid apoptosis are essential in placental implantation.Epigenetic modification affects gene activity by changing the accessibility of genomic DNA to transcription factors.Epigenetic regulation of placenta refers to the regulation of gene expression through DNA methylation,histone modification and RNA regulation without changing DNA nucleotide sequence,thus affecting the synthesis of corresponding proteins in placenta to regulate the development of placenta.DNA methylation is the most widely studied and characteristic epigenetic modification mode.The abnormal epigenetic mode in human placenta,especially the change of DNA methylation status in the promoter region of susceptible genes,is related to the pathophysiology of various pregnance-related complications.The results of animal experiments showed that bisphenol A caused an increase in the methylation level of Wnt2 in the placenta of mice,resulting in the down regulation of Wnt2 expression and the occurrence of preeclampsia syndrome in mice.We speculate that during the pathogenesis of preeclampsia,the abnormal epigenetic regulation of Wnt2 gene leads to changes in gene expression,which then affects the biological behavior of trophoblast cells,and may be involved in the pathogenesis of preeclampsia.This study aims to explore the possible molecular mechanism of Wnt2 gene in the pathogenesis of preeclampsia by studying the expression and regulatory mechanism of Wnt2 gene in placental tissues,and to provide a new theoretical basis tor the early diagnosis and treatment of preeclampsia.Part Ⅰ Expression of Wnt2 gene in placenta of preeclampsia patientsObjective:The expression of Wnt2 gene mRNA and protein in the placental tissues of early-onset severe preeclampsia patients,late-onset severe preeclampsia patients and normal women with late pregnancy were detected to investigate the role of Wnt2 gene in the pathogenesis of preeclampsia.Methods:1.ratients studiedThis study was conducted in the Departnent of Gynaecology and Obstetrics,Binzhou Medical University Hospital,Binzhou,Shandong,China,between January 2018 and December 2018.30 patients with early-onset severe preeclampsia were treated as early-onset severe preeclampsia group(ZPE),30 patients with late-onset severe preeclampsia were treated as late-onset severe preeclampsia group(WPE),and 30 normal late pregnancy women were treated as control group(NC).All pregnant women in the study were delivered by cesarean section.2.MethodsThe mRNA expression of Wnt2 gene in placenta of early-onset severe preeclampsia patients,late-onset severe preeclampsia patients and normal women of late pregnancy was detected by qRT-PCR.And Wnt2 protein expression was detected by Western blot.Results:1.There were no statistically significant differences in mean age,body mass index and gestational time between the early-onset severe preeclampsia group(ZPE),the late-onset severe preeclampsia group(WPE)and the control group(P>0.05).There were statistically significant differences in systolic blood pressure,diastolic blood pressure,urinary protein content,gestational age,neonatal birth weight and Apgar score of 1min between the control group and the early-onset severe preeclampsiagroup(ZPE)(P<0.05).There were statistically significant differences in systolic blood pressure,diastolic blood pressure,urinary protein content,gestational age,neonatal birth weight and neonatal Apgar score of lmin between the control group and the late-onset severe preeclampsia group(WPE)(P<0.05).There were no statistically significant differences in systolic blood pressure,diastolic blood pressure and urinary protein content between the early-onset severe preeclampsia group(ZPE)and the late-onset severe preeclampsia group(WPE)(P>0.05).There were significant differences in neonatal Apgar score of lmin(P<0.05).2.qRT-PCR results showed that the relative expression level of Wnt2 mRNA in the placenta of patients with early-onset severe preeclampsia was significantly lower than that in the control group(P<0.05).The relative expression level of Wnt2 mRNA in placenta of patients with late-onset severe preeclampsia was significantly lower than that in the control group(P<0.05).The relative expression level of Wnt2 mRNA in placenta of patients with early-onset severe preeclampsia group(ZPE)was significantly lower than that in patients with late-onset severe preeclampsia group(WPE),and the difference was statistically significant(P<0.05).3.Western blot results showed that the expression of Wnt2 protein in placenta of patients with early-onset severe preeclampsia was significantly lower than that in the control group,with statistically significant difference(P<0.05).The expression level of Wnt2 protein in placenta of patients with late-onset severe preeclampsia was significantly lower than that in the control group(P<0.05).The expression level of Wnt2 protein in placenta of patients with early-onset severe preeclampsia was significantly lower than that in patients with late-onset severe preeclampsia(P<0.05).4.Immunohistochemistry results showed that Wnt2 protein was mainly expressed in the membrane and cytoplasm of villous trophoblast cells.The expression level of Wnt2 protein in placenta of patients with early-onset severe preeclampsia was significantly lower than that in the control group(P＜0.05).The expression level of Wnt2 protein in placenta of patients with late-onset severe preeclampsia was significantly lower than that in the control group(P<0.05).There was no significant difference in the expression of Wnt2 protein in placenta between the early-onset severe preeclampsia group(ZPE)and the late-onset severe preeclampsia group(WPE)(P>0.05).Conclusion:1.Wnt2 mRNA and protein were expressed in the placenta of normal women in the third trimester and patients with severe preeclampsia.2.The expression levels of Wnt2 gene mRNA and protein were highest in the placenta of normal women in the third trimester,followed by late-onset severe preeclampsia patients,and lowest in the placenta of early-onset severe preeclampsia patients.3.Wnt2 protein was mainly expressed in the membrane and cytoplasm of placental villous trophoblast cells.Part Ⅱ Effects of Wnt2 gene on biological behavior of human trophoblast cellsObjective;To investigate the role of Wnt2 on the biological behavior and apoptosis of trophoblast cells in preeclampsia,Wnt2 gene was overexpressed and knocked down in HTR8 cells by transfection.So as to provide a new idea for the early diagnosis and effective treatment of preeclampsia.Methods:CCK8 assay,colony-forming assay,Transwell assays,scratch wound healing assay and flow cytometry were performed to assess cell biological behaviors(cell proliferation,clonogenesis,migration,invasion and apoptosis)of HTR8 cells in the Wnt2 gene knockdown(KD)group and the WntZ gene overexpression(OE)group.To study the changes of apoptosis related proteins and cell migration and invasion related proteins in the Wnt2 gene knockdown(KD)group and the Wnt2 gene overexpression(OE)group.Results:1.The results of CCK8 assay showed that,compared to NC group,the HTR8 cell viability in OE group was significantly increased,but was markedly decreased in KD group(P<0.05).2.The colony-fonning assay confirmed the colony-forming ability of HTR8 cells in OE group was significantly stronger than NC group,while that in KD group was obviously weaker than NC group(P<0.05).These data indicated that overexpression of Wnt2 promoted trophoblast cell proliferation,whereas knockdown of Wnt2 had opposite effects.3.The results of flow cytometry showed that compared with the NC group,knockdown Wnt2 promoted apoptosis of HTR8 cells,while overexpression of Wnt2 inhibited apoptosis(P<0.05).4.The results of Transwell migration assay showed that the migration number of IITR8 cells in OE group was signiticantly increased compared to NC group,while distinctly decreased in KD group(P＜0.05).5.Similar results were also verified by Transwell invasion assay that the invasion number of HTR8 cells in OE group was significantly increased compared to NC group,while distinctly decreased in KD group(P<0.05).6.The results of scratch wound healing assay showed that the dosed wound area of HTR8 cells in OE group was remarkably larger than that of NC group,while that in KD group was dramatically smaller than NC group(P<0.05).7.Western blot assay was performed to detect the expression of epithelial nesenchymal transition markers.The results showed that overexpression of Wnt2 in OE group resulted in the down-regulation of Bcl-2 expression and up-regulation of the expression of Cleaved-Caspases3 and Bax,whereas knockdown of Wnt2 in KD group leaded to the opposite changes of expression in these proteins(P<0.05).8.Overexpression of Wnt2 in OE group resulted in the down-regulation of E-cadherin expression and up-regulation of the expressioi of N-cadherin,Vimentin and 13-Catenin?whereas knockdown of Wnt2 in KD group leaded to the opposite changes of expression in these proteins(P<0.05).Conclusion:1.Wnt2 gene promoted the proliferation,migration and cloning ability of human chorionic trophoblast cells,and inhibited the apoptosis of trophoblast cells.2.The possible mechanism that the down-regulated expression of Wnt2 gene affects the biological behavior of trophoblast cells was as follows:the down-regulation of Wnt2 gene inhibited the classical Wnt signal transduction pathway,leading to the reduced migration and invasion ability of trophoblast cells.The down-regulation of Wnt2 gene promoted the caspase-mediated apoptosis and caused the increase of trophoblast cell apoptosis.3.The down-regulation of Wnt2 gene was involved in the pathogenesis of preeclampsia by affecting the biological behavior of trophoblast cells,thereby leading to placenta formation defects and dysfunction.Part Ⅲ Epigenetic regulation of Wnt2 gene in thepathogenesis of preeclampsiaObjective:The DNA methylation degree in the promoter region of Wnt2 gene in theplacenta of normal late pregnancy women,early-onset severe preeclampsia and late-onset preeclampsia was detected.To explore the mechanism of reduced Wnt2 expression inducing preeclampsia and provide new ideas for early prediction and clinical treatment of preeclampsia.Methods:1.Patients studiedThis study was performed in the Department of Gynaecology and Obstetrics of Binzhou Medical University Hospital,Binzhou,Shandong,China,between January 2018 and December 2018.30 patients with early-onset severe preeclampsia were treated as early-onset severe preeclampsia group(ZPE),30 patients with late-onset severe preeclampsia were treated as late-onset severe preeclampsia group(WPE)(WPE),and 30 normal late pregnancy women were treated as control group(NC).All pregnant women in the study were delivered by cesarean section.2.MethodsMethylation-specific polymerase chain reaction was used to detect the DNA methylation in the Wnt2 gene promoter region in placenta of early-onset severe preeclampsia patients,late-onset severe preeclampsia patients and normal women with late pregnancy.Results:1.The Wnt2 gene was partial methylated in the placentas of early-onset severe-preeclampsia patients,late-onset severe preeclampsia patients and normal women with late pregnancy.2.The level of DNA methylation in the promoter region of Wnt2 gene in placental tissues of the early-onset severe preeclampsia group(ZPE)was higher than that of the control group,with statistically significant difference(P<0.05).The level of DNA methylation in the promoter region of Wnt2 gene in placental tissues of the late-onset severe preeclampsia group(WPE)was higher than that of the control group,and the difference was statistically significant(P<0.05).However,there was no statistically significant difference in DNA methylation in the Wnt2 promoter region between the early-onset severe preeclampsia group(ZPE)and the late-onset severe preeclampsia group(WPE)(P>0.05).Conclusion:1.Incomplete DNA methylation in the Wnt2 promoter region was found in the placentas of normal women in the third trimester,early-onset severe preeclampsia and late-onset severe preeclampsia.2.Compared with normal pregnant women,DNA methylation in the Wnt2 promoter region in placenta of patients with early-onset severe preeclampsia and late-onset severe preeclampsia was significantly increased.3.Intervention of DNA methylation state in Wnt2 promoter region can regulate the expression of Wnt2.Wnt2 gene is expected to be a new point for early diagnosis and treatment of preeclampsia.