The Study Of The YAP Expression In Preeclampsia And The Mechanism Of Trophoblast Function

Objective: Preeclampsia(PE)is a kind of pregnancy-induced hypertension(Hypertension).It is an idiopathic pregnancy disorder whose main clinical manifestation is hypertension and proteinuria after 20 weeks of gestation.It even causes multiple organ injury and serious Affect maternal and child health,maternal and perinatal mortality is an important reason.The incidence of the disease remains high,so far the pathogenesis is unknown,the etiology of many,mainly uterine spiral arterial insufficiency(commonly known as "placenta asymptomatic"),inflammatory immune activation,vascular endothelial cell damage(including inflammatory mediators,Oxidative stress,etc.),genetic and nutritional deficiencies,insulin resistance and the like.Including uterine spiral arterial remodeling disorders and vascular endothelial cell damage to a certain extent and trophoblast behavior changes are closely related.Indepth study of factors that affect the biological function of trophoblast cells and clarify the relevant regulatory mechanism,thereby preventing the occurrence of disease development and reduce the incidence of perinatal disease.In recent years,studies have shown that Hippo pathway regulates organ size by controlling the balance between proliferation and apoptosis.In animal models,inhibition of Hippo signaling leads to tumor cell overgrowth and tumorigenesis.Thus Hippo pathway can serve as a potential node for tumor suppressor and therapeutic intervention.YAP,a transcriptional coactivator,plays a central role in the Hippo signaling pathway.The activation of Hippo pathway in a series of kinases leads to phosphorylation of YAP in the cytoplasm.Non-phosphorylated YAP can enter into the nucleus to participate in a series of transcription factor activation processes such as TEADs,SMAD,RUNX and CTGF.YAP gene is also involved in a variety of tumor biological behaviors such as tumor stem cell maintenance,epithelial mesenchymal transition(EMT),migration and invasion and chemoresistance.Highly expressed YAP plays an important role in tumorigenesis,development and relapse.Trophoblast cells play a tumor-like role in embryogenesis.However,the current mechanisms of how YAP regulates trophoblastic differentiation and promote trophoblast implantation and trophoblast function remain unclear.Methods: Part I: the expression of YAP in placenta 30 cases of early pregnancy villus(placenta in early pregnancy),30 cases of fullterm placenta,30 cases of preeclampsia placenta,early pregnant pregnancy group and full term pregnancy group,healthy pregnant women without symptoms,signs and medical history of threatened abortion,These pregnant women did not receive antiinflammatory treatment,no infection,chorionitis and other complications.Real-time quantitative PCR method was used to detect the expression of YAP in early pregnancy,full term pregnancy placenta and preeclampsia placenta.Immunohistochemistry was used to detect the expression of YAP in placenta tissue.Part II: Effects and Mechanisms of YAP on Trophoblast Infiltration,Apoptosis and Proliferation Biological Functions HTR-8 / SVneo trophoblasts were cultured in vitro,and transfected into HTR-8 / SVneo cells by lipo 3000-mediated transfection of YAP-overexpressed plasmid and YAP siRNA.QRT-PCR and Western Blot were used to detect the protein level and protein expression.Transwell method was used to observe the change of HTR-8 / SVneo infiltration.The apoptosis of HTR-8 / SVneo was detected by flow cytometry.The proliferation of HTR-8 / SVneo was detected by CCK-8.The mRNA and protein expression of CDX2 downstream of YAP were detected by qRT-PCR and Western Blot,and the further study on the biological function of Hippo-YAP via CDX2 on trophoblast cells.Results: Part I: We use qRT-PCR and Western Blot method to detect YAP mRNA and protein expression in early pregnancy villus,full term placenta and preeclampsia placenta.Placenta and early pregnancy expression levels.The results showed that in the preeclampsia placenta YAP mRNA and protein levels were reduced in the first trimester villi YAP mRNA and protein levels were increased.We use immunohistochemical techniques to further determine the expression of YAP in placenta tissue parts found YAP expression in syncytiotrophoblasts,YAP in preeclampsia placenta tissue expression levels are relatively low.The results suggest that YAP is likely to participate in trophoblast biological behavior,causing preeclampsia incidence.Part II: We constructed YAP overexpression / silencing expression system by transfection technique to further study the function of trophoblast cells.The expression of YAP in HTR-8 / SVneo cells transfected with pc DNA3.1-YAP plasmid was significantly up-regulated <0.01).The expression level of YAP in HTR-8 / SVneo cells transfected into si-YAP was significantly down-regulated(P <0.01),but there was no significant difference between the empty vector plasmid group and the untreated siRNA group difference.The number of cells in the overexpression group and the control group was 201.5 ± 4.272 and 104.8 ± 2.689,respectively.The number of cells in the si-YAP and control groups was 48.50 ± 1.323 and 96.50 ± 2.500,respectively(P < 0.01,the difference was statistically significant).YAP protein can promote the infiltration capacity of HTR-8 / SVneo cells.We used flow cytometry to detect cell apoptosis,the results suggest that: over-expression and control group apoptosis rates were 13.48 ± 0.4193,23.61 ± 0.7363,si-YAP and control group apoptosis rates were 36.40 ± 0.6616,25.95 ± 0.9233,(P <0.01,the difference was statistically significant).In conclusion,YAP protein can inhibit the apoptosis of HTR-8 / SVneo cells.We detected the proliferation of HTR-8 / SVneo cells by CCK-8 assay.The proliferation of HTR-8 / SVneo cells was detected at 24 h,48h and 72 h after plating,respectively.The upregulation / down-regulation of YAP had no significant effect on the proliferation of HTR-8 / SVneo cells.QRT-PCR and Western Blot were used to detect the mRNA and protein expression of CDX2 after up-regulation / down-regulation of YAP.The results showed that upregulation of YAP expression could significantly increase the expression of CDX2 mRNA and protein,and down-regulation of YAP expression could significantly decrease the expression of CDX2 mRNA and protein.Therefore,we hypothesize that YAP is likely to affect trophoblast invasion and apoptosis through CDX2 and participate in the pathogenesis of preeclampsia.Conclusion: 1.YAP mRNA and protein levels in preeclampsia placenta significantly decreased,YAP most likely involved in the pathogenesis of preeclampsia.2.YAP involved in trophoblast biological behavior.3.YAP can enhance the infiltration capacity of HTR-8 / SVneo and inhibit the apoptosis of HTR-8 / SVneo,which has no effect on the proliferation of HTR-8 / SVneo.4.YAP is likely to affect trophoblastic invasion and apoptosis through CDX2 and participate in the pathogenesis of preeclampsia.