| Preeclampsia (PE) is a common, pregnancy-specific disease that belongs to the family of hypertensive disorders in pregnancy and is characterized by new onset of hypertension and proteinuria after20weeks of gestation. PE is a major contributor to maternal and fetal morbidity and mortality. PE affects5-8%of pregnancies internationally, including9.4%of pregnancies in China. However, the precise mechanisms of PE pathogenesis remain unknown.The literature consensus indicates that the placenta is the central organ in the pathogenesis of PE, and clinical investigations have shown that the disease remits dramatically after the placental expulsion. Therefore, it is a widely accepted hypothesis that placenta dysfunction may contribute to the process of PE. Trophoblasts are the primary cell types found in the placenta, and they are referred to as false malignant cells because their biological behavior resembles that of highly invasive tumor cells. The normal differentiation, proliferation, apoptosis migration and invasion ability of trophoblasts are crucial to the subsequent embryo implantation and placentation. However, some of the placental abnormalities, including deficient implantation, abnormal cytotrophoblast invasion of spiral arterioles, and improper placental vascular development, are believed to lead to PE.Signaling by the Wnt family is one of the critical mechanisms that directs cell proliferation, migration, polarity, and tissue homeostasis. The canonical Wnt/β- catenin signaling is activated when a Wnt ligand binds to the Frizzled receptors and their co-receptors. The signal is then transmitted into the cytoplasm by a series of cellular factors, and these events lead to the stabilization of β-catenin in the cytoplasm. Consequently,β-catenin accumulates and travels into the nucleus to form complexes with T cell-specific factors (TCFs)/lymphoid enhancer-binding factor1(LEF-1), which in turn activate target gene expression. Wntl is an initiating factor of the Wnt signaling pathway, which could give rise to apoptosis inhibition, cell invasion and angiogenesis in tumor formation by activating the Wnt/β-catenin canonical pathway.β-catenin is the primary Wnt effector, which serves as a coactivator through its ability to recruit the components that promote chromatin remodeling and transcriptional initiation/elongation. Dickkopf-1(DKK1) is a secreted glycoprotein that can antagonize the canonical Wnt signaling pathway, and this cascade influences numerous biological processes. Accumulating evidence suggests that Wnt signaling has been identified as an essential pathway that promotes endometrial function, decidualization, trophoblast differentiation and invasion. However, the potential roles of the Wnt pathway in the pathogenesis of severe PE have not been well elucidated. The objectives of the current study were to detect the expression of Wntl, DKKland β-catenin in the placenta affected with severe preeclampsia (PE) in order to understand their potential roles in the pathogenesis of severe PE.ObjectiveThe objectives of this study were to detect the mRNA and protein expression levels of Wntl, DKKland P-catenin in the placenta using Real-time polymerase chain reaction (real-time-PCR) and western blot, evaluate their locations by immunohistochemistry (IHC), in order to understand their potential roles in the pathogenesis of severe PE. Materials and methods1MaterialsSixty pregnant women who had undergone cesarean section at The Third Affiliated Hospital of Zhengzhou University, from January2010to January2012, were enrolled in this study. This sample included30healthy pregnant women, who constituted the control group, and30preeclamptic women, who were classified as the severe PE group. In China, the diagnosis of PE is based on the definitions outlined in the seventh edition of "Obstetrics and Gynecology", which was published by the People’s Medical Publishing House. Subjects with diabetes mellitus, chronic hypertension, renal disease, polycystic ovarian syndrome, multiple gestations and fetal malformations were excluded from this study.2MethodsThe placental biopsies were collected from the maternal aspect of the placenta within15min of delivery. Five small separate biopsies (2cm x2cm x1cm) were excised from the placental center, as well as from each quadrant (3,6,9, and12o’clock of the placenta) to avoid sampling bias. Biopsies were not collected from the placental periphery or areas of calcification and necrosis. For the current study involving the analysis of placental RNA and protein levels, the biopsies were flash frozen in liquid nitrogen and stored at-80℃until the tissue was processed. Tissue was fixed in10%formalin for immunohistochemical assays.3Statistics analysisAll statistical analyses were performed using SPSS17.0software. Quantitative data are presented as mean±standard deviation (SD). Comparison of two groups was performed using either unpaired t test (for parametric data) or the Mann-Whitney U-test (for non-parametric data). The differences in enumeration data were detected with the x2test. The2-△△CT method was used to analyze the relative gene expression from real time PCR data. Differences were considered to be statistically significant when P＜0.05.Results1The demographic characteristics of the two groupsThe Maternal age of severe PE group and normal control group were29.17±5.48and30.73±3.35, respectively. Maternal BMI of severe PE group and normal control group were29.50±3.87,29.06±3.68respectively, The maternal age and maternal BMI were no significant differences between the two groups (P>0.05); however, the gestational age of severe PE group was35.35±2.69, which was significant lower than the normal control group38.19±1.76(P＜0.05). The neonatal weight was significantly decreased in severe PE group2240.00±729.71,compared to those from the normal control group3248.37±529.03(P＜0.05).2The Wnt1,DKK1, β-catenin mRNA expression in placenta of the severe PE and normal control group2.1The Wntl mRNA expression in placenta of the severe PE and normal control groupOur results indicated that Wntl mRNA expression could be detected in both the severe PE and normal control groups. The Wntl mRNA expression in the severe PE group was decreased, compared with the normal control group (P＜0.05).2.2The DKKl mRNA expression in placenta of the severe PE and normal control groupThe DKKl mRNA expression could be detected in both the two groups. The DKK1mRNA expression of severe PE group was significantly increased (P＜0.05). 2.3The β-catenin mRNA expression in placenta of the severe PE and normal control groupThe β-catenin mRNA expression could be detected in both the two groups. In the severe PE group, the β-catein mRNA expression was decreased, compared with the normal control group (P＜0.05).3Localization of Wntl, DKK1and β-catenin protein expression in the placenta during the third trimesterWe observed that the immunostaining for the Wntl, DKK1and β-catenin proteins was observed in syncytiotrophoblasts and the extravillous trophoblasts (EVT). Our results indicated that the staining intensity of Wntl and β-catenin in the placental tissue of the severe PE group was weaker than the control group. However, the staining intensity of DKK1showed the opposite pattern, with greater staining in the control group.4The differential expression of Wntl, DKKland β-catenin protein in normal and severe PE group4.1The Wntl protein expression in placenta of the severe PE and normal control groupThe Wntl protein expression was significantly decreased in the severe PE group compared with the normal control group(P＜0.05).4.2The DKK1protein expression in placenta of the severe PE and normal control groupThe DKK1expression was significantly increased in the severe PE group compared with the normal control group (P＜0.05). 43The β-catenin protein expression in placenta of the severe PE and normal control groupCompared with the normal control group, the β-catenin expression in the severe PE group was significantly decreased (P＜0.05).ConclusionThe results of this study indicated that Wntl, DKK1and β-catenin and are expressed in human third trimester placentas, decreased Wntl and β-catenin expression, as well as DKK1over-expression might be involved in the process of the pathogenesis of PE. |
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