DIA-based On Proteomics Of Pernicious Placenta Previa With Placenta Accrete And Research Of Zeb-1 In The Placenta Accrete

Introduction: Morbidly adherent placenta(MAP)is a group of abnormal placental attachment to the uterus.It can be divided into abnormal placenta attachment sites,such as placenta previa(PP)and abnormal placenta attachment depth,such as placenta accreta spectrum(PAS).Placental hyperplasia refers to the pathological condition of abnormal placental chorion implantation into uterine wall caused by partial or total absence of decidual basal layer.It is now the main cause of postpartum hemorrhage,which can lead to various diseases of mothers and newborns.Placental villi can not only invade the myometrium of uterus,but also penetrate the uterine wall into the outer serosa or even the bladder,so that the placenta can not be removed from the uterus.Studies have shown that the incidence of placental hyperplasia has increased significantly over the past 50 years.It is difficult for placenta implantation patients to dissect the placenta completely during delivery,which can easily lead to intrapartum and postpartum hemorrhage.The main cause of emergency perinatal hysterectomy is the large average amount of bleeding.The maternal mortality rate caused by placenta implantation is as high as 7-10%,which is a serious obstetric complication.The first choice of prenatal diagnosis is ultrsound examination.Prenatal ultrasound examination can evaluate the degree of placenta implantation and,and make adequate preoperative preparation,which can reduce intraoperative bleeding and serious complications.The molecular basis of placental implantation has not been elucidated,but it may involve abnormal protein expression and signal transduction between maternal decidua and trophoblast.The molecular basis of invasive placenta formation has not been elucidated,but it may involve abnormal protein expression and signal transduction between maternal decidua and trophoblast.Proteomic studies using disease tissues as experimental materials can not only provide new specific candidate markers for early diagnosis of diseases,but also find new specific candidate antigens for vaccine development,and provide clues for understanding pathogenesis.Zinc finger E-box-binding protein(Zeb)plays an important role in cell differentiation and embryonic development.Zeb1 is reported to be closely related to biological processes such as invasion and metastasis of tumors.Although the invasion mechanism of embryo implantation into endometrium is similiar to that of tumors,little research has been done on Zeb1 in placenta implantation.Objective: Explore the situation of proteomic differential proteins in pernicious placenta previa with placenta accrete,the expression of Zeb1 in placenta tissue of placenta accreta,and the effect of Zeb1 on the cellular biological function of HTR-8/SVneo and HUVECs cells.Methods: In the first part,we visited 150 cases of pernicious placenta previa from November 2015 to November 2017 in ourhospital.Prenatal ultrasound(2-D+3-D color Doppler+3-D power Doppler)was performed within 72 hours before operation,and maternal blood and placental tissue samples were collected.A retrospective analysis of three-dimensional power Doppler ultrasound in predicting pernicious placenta previa implantation and the degree of danger was made.Protein quantitative DIA technique was used to identify and screen differentially expressed proteins of pernicious placenta previa with placenta accrete and and VFI > 20.The second part: study the expression of Zeb1 in placental tissues of placenta accreta.Experimental group:(1)placenta accreta group: 20 placental tissues at placenta implantation site diagnosed by preoperative ultrasound VFI > 20 and pathology after operation;(2)Non-accreta group: 20 placental tissues with preoperative ultrasound VFI < 10 and no placenta accreta during operation;(3)Normal control group: single and late pregnancy with the history of cesarean section,placental tissue of 20 patients who terminated pregnancy by cesarean section.Methods: the expression of Zeb1,VEGF and E-cad in three groups of tissues was analyzed by immunohistochemical staining.The expression of Zeb1,E-cad,N-cad and VEGF in three groups was analyzed by Western blot and real-time PCR.The third part: HTR-8/SVneo human choriotrophoblast cells were selected as the research object.Zeb1 was over-expressed and silenced in the cells respectively.The effects of Zeb1 on proliferation and apoptosis of HTR-8/SVneo human choriotrophoblast cells were analyzed by MTT,flow cytometry and AV-PI staining.HTR-8/SVneo human choriotrophoblast cells were selected as the research object.Zeb1 was overexpressed and silenced in the cells respectively.The effect of Zeb1 on the migration ability of HTR-8/SVneo human choriotrophoblast cells was analyzed by Transwell technique.The protein content and RNA content of Zeb1 in HTR-8/SVneo human choriotrophoblast cells were detected by Western blot and real-time PCR,respectively,including Zeb1,E-cad,N-cad,VEGF,Cyclin D1 and bcl-2.Human umbilical vein endothelial cells(HUVECs)were selected as the research object.Zeb1 was overexpressed and silenced in HUVECs.MTT,flow cytometry and AV-PI staining were used to analyze the effects of Zeb1 on proliferation and apoptosis of HUVECs human umbilical vein endothelial cells.Human umbilical vein endothelial cells(HUVECs)were selected as the research object.Zeb1 was overexpressed and silenced in HUVECs.The effect of Zeb1 on migration ability of HUVECs human umbilical vein endothelial cells was analyzed by Transwell technique.Western blot and real-time PCR were used to detect the protein and RNA contents of Zeb1,E-cad,N-cad,VEGF,Cyclin D1 and Bcl-2 in HUVECs human umbilical vein endothelial cells.Statistical Analysis: Data are expressed as means ± SD and were analyzed with SPSS software.Differences between group means were calculated by one-way analysis of variance(ANOVA)followed by Turkey’s multiple comparison tests.Results were considered statistically significant when P< 0.05.Results: 1.VFI in 3D-PDU as a quantitative index to evaluate the degree of pernicious placenta previa placenta implantation,VFI < 10 suggested no significant placenta accrete was predicted,VFI > 20 suggested that placenta increte or pencrete was more likely;2.160 proteins were expressed differently between pernicious placenta previa with accrete and non-accrete.38 proteins were up-regulated and 122 proteins were down-regulated,which provided a basis for predicting and related research of pernicious placenta previa with placenta accrete;3.Zeb1,N-cad and VEGF were most expressed in pernicious placenta accreta tissues and least expressed in normal control tissues.The expression of E-cad was the lowest in pernicous placenta accreta tissues and the highest in normal control tissues.4.Zeb1 can promote HTR-8/SVneo and HUVECs cell proliferation,inhibit cell apoptosis and promote cell migration.5.Western blot and real-time PCR showed that over-expression of Zeb1 could up-regulate the contents of Zeb1,N-cad,VEGF,Cyclin D1 and bcl-2,and down-regulate the expressions of E-cad.Conlusions: 1.Three-dimensional power Doppler ultrasound score can assess the dangerous degree of placenta implantation.2.Zeb1 plays an important role in the pathogenic attachment of placenta.3.Zeb1 regulates the proliferation,apoptosis and migration of HTR-8/SVneo and HUVECs.