The Detection Of Wnt2b, WIF-1and E-cadherin Expression In Preeclampsia By Placenta Tissue Microarray

Preeclampsia (PE) is a placental syndrome becoming clinically apparent in laterpregnancy with maternal hypertension, proteinuria that affecting5-8%of pregnanciesworldwide, and9.4%in china. It is a major obstetrical problem leading to thesignificant maternal and fetal morbidity and mortality. There are numerous etiologicaltheories regarding PE, but its precise pathogenesis has not been elucidated yet.Placenta, the direct interface between the fetus and the mother during pregnancy, mayplay a central or initiating role in this disorder, based on the fact that the clinicalsymptoms of PE rapidly vanish after delivery, including the removal of the placenta.Trophoblasts are the dominating cell types of the placenta, which has the ability ofdifferentiation, proliferation, migration and invasion that is crucial to the placentationand embryo implantation. Studies have demonstrated that the incomplete trophoblastassociated invasion and arterial remodeling are associated with PE.The Wnt pathway represents the key signaling networks involved in cellproliferation, migration and differentiation. The aberrant Wnt signaling is associatedwith several forms of cancers. Wnt2b/wnt13is one of the initial factors of canonical Wnt pathway. It affects tumors progression by inhibiting cell apoptosis, promotingcell invasion and angiogenesis through the activation of Wnt/β-catenin pathway. Wntinhibitory factor-1(WIF-1) is a member of secreted frizzled-related proteins. It exertsits function by binding directly to Wnt proteins and inhibiting their ability to interactwith Frizzled. The re-expression of WIF-1may therefore result in the downregulationof the Wnt pathway and the inhibition of the cancer cell growth. E-cadherin is animportant cell adhesion and signal transduction molecules on the cell membrane. Itantagonizes the Wnt pathway by integrating withβ-catenin. Now accumulating studieshave demonstrated that Wnt pathway involved in regulating trophoblasts invasion anddifferentiation, but little information was known about the pathogenesis of PE. Theconstruction and validation of the placental tissue microarray provided ahigh-efficient tool for the array-based protein expression survey in PE study. In thisstudy, we employed immunohistochemistry (IHC) to examine the protein level ofWnt2b, WIF-1and E-cad on placenta villous cytotrophoblast (VCT) and extravillouscytotrophoblast (EVCT) tissue microarray, and Real-time PCR to examine the mRNAlevel of Wnt2b, WIF-1and E-cad in placentas of severe PE (sPE) and normal controls,in order to investigate the potential role of Wnt signaling in the pathogenesis of PE.ObjectiveThe objective of this study were to detect the protein level of Wnt2b, WIF-1andE-cad on VCT and EVCT tissue microarrays by using immunohistochemistry (IHC),and the mRNA level of Wnt2b, WIF-1and E-cad in placentas of sPE and normalcontrols by using Real-time PCR, in order to investigate the potential role of Wntsignaling in the pathogenesis of PE. Materials and Methods1Materials1.1Materials of Real-time PCRThirty preeclamptic women and thirty normal pregnant women who hadundergone cesarean section were recruited from the Third Affiliated Hospital ofZhengzhou University between December2011and December2012for mRNAdetection.1.2Materials of Placenta Tissue MicroarrayHenan translational medicine engineering laboratory for Maternal and children’shealth selected80preeclampsia and58normal placental specimens for constructingplacenta VCT and EVCT TMA.1.3The Diagnosis Criteria of Severe PreeclampsiaThe diagnosis criteria of severe PE were strictly based on the AmericanCongress of Obstetricians and Gynecologists criteria (ACOG,2002) and the seventhedition of “Obstetrics and Gynecology” which published by the People’s MedicalPublishing House. The normal pregnancy was defined as the pregnancy characterizedby normal blood pressure values (<140/90mmHg) and negative proteinuria. Patientswith chronic hypertension, diabetes mellitus, renal disease, polycystic ovariansyndrome, fetal malformations and multiple gestations were excluded from this study.2The information of VCT and EVCT tissue microarraysEach target area was punched triplicate exactly to increase the consistency of theresults. We used cytoplasmic staining for cytokeratin, vimentin and HLA-G to verifythe placental VCT TMA and EVCT TMA, the results had shown that the cytoplasmicstaining all accorded with the characteristics of VCT and EVCT tissues, which meansthe TMAs were successfully constructed. Besides, Henan translational medicineengineering laboratory for Maternal and children’s health offered the placental VCTTMA and EVCT TMA for this study.The H&E staining had shown that98.97%(97/98) VCT TMA samples and86.36%(76/88) EVCT TMA samples retained target tissues, which means we could analyze56PE (incuding48severe PE,8mild PE;41early-onset PE,15late-onset PE)and42normal control samples on VCT TMA,47PE (incuding44severe PE,3mildPE;38early-onset PE,9late-onset PE) and29normal control placenta samples onEVCT TMA. Owing to the less sample number of mild PE and late-onset PE, we putmore emphasis on the protein analysis of severe PE and early-onset PE.3Methods3.1The location and expression of Wnt2b, WIF-1and E-cad in theplacentas by immunohistochemistryThe paraffin VCT TMA and EVCT TMA blocks were cut into4μm sections forimmunohistochemistry (IHC) analysis. The expression and location of Wnt2b, WIF-1and E-cad proteins in placentas from PE and normal controls were detected by IHC.3.2The mRNA level of Wnt2b, WIF-1and E-cad in the placentas withsPE and normal pregnancies by Real-time PCRTotal RNA was extracted from the placentas by using Trizol Reagent and thenreverse transcribed to cDNA. We employed Real-time PCR on an AppliedBiosystems7500with the Ultra SYBR Mixture to examine the mRNA level ofWnt2b, WIF-1and E-cad in the Placentas. Human glyceraldehydes-3-phosphatedehydrogenase (GAPDH) was used as the internal control. The mRNA level ofWnt2b, WIF-1and E-cad in the Placentas was calculated by2-ΔΔCTmethod.4Statistics analysisAll statistical analysis was performed using SPSS version17.0software.Quantitative data are presented as the mean±standard deviation (SD). Comparison oftwo groups was performed using either unpaired t test (for parametric data) or theMann–Whitney U-test (for non-parametric data). The differences in enumeration datawere detected with the χ2test. A P <0.05was considered statistically significant. Results1The demographic characteristics of the two groupsThe maternal age, maternal BMI (body mass index) were no significantdifferences between sPE group and normal control group. However, the systolicpressure and diastolic pressure, the degree of proteinuria of sPE group weresignificantly higher than the normal control group (P<0.05); The maternal gestationalage (week), the neonatal birth weight of sPE group were significantly lower than thenormal control group (P <0.05).2The mRNA level of Wnt2b, WIF-1and E-cad in the placentaswith sPE and normal control groupOur results indicated that Wnt2b, WIF-1and E-cad mRNA expression could bedetected both in the sPE and normal control group. Compared with the control group,the mRNA level of Wnt2b was downregulated, while the mRNA level of E-cad wasincreased in the sPE group (P <0.05).However, there was no significant differencein WIF-1expression between the two groups (P=0.66).3Location of Wnt2b, WIF-1and E-cad of PE group and normalcontrol group on VCT and EVCT TMA.The immunostaining of the Wnt2b, WIF-1and E-cad proteins were all foundmainly in the villous syncytiotrophoblast and the EVCT. The Wnt2b proteins werefound in the cytomembrane and cytoplasm, the WIF-1proteins were found in thecytoplasm and extracellular matrix, the E-cad proteins were found mainly in thecytomembrane. The staining intensity of Wnt2b on VCT and EVCT TMA of the PEgroup was weaker than the normal control group (P <0.001). The staining intensityof WIF-1, E-cad on VCT and EVCT TMA of the PE group were greater than thenormal control group (P <0.001). Compared with the normal control group, thestaining intensity of Wnt2b were weaker in the sPE group, early onset PE group (P < 0.05), while the staining intensity of WIF-1, E-cad was stronger (P <0.05).ConclusionsTaken together, the result of this study indicated that Wnt2b, WIF-1and E-cadare all expressed in human third trimester placentas, and the decreased expression ofWnt2b, as well as the over-expression of WIF-1and E-cad may be associated withthe pathogenesis of PE.