Study About Methylation And Transcription Levels Of WNT2 And DKK1 In Placentas Associated With Early-onset Preeclampsia

Preeclampsia(PE)is a pregnancy-specific disorder,with hypertension and proteinuria arising in the second or third trimester of pregnancy,and may be combined with systemic complications including kidney failure,liver dysfunction and neurological clinical manifestations such as seizures.PE is the one of the principal causes of an increase of maternal and fetal mortality and morbidity,and may lead to fetal intrauterine growth restriction and preterm birth.The onset of this pregnancy complication is affected by the combination of genetic and environmental factors.At present,the exact pathogenesis of PE remains unclear,its occurrence is the result of various pathogenesis including inadequate uterine spiral artery remodeling,placental oxidative stress,vascular endothelial cell dysfunction and abnormal maternal inflammation response.PE can't be diagnosed until the third trimester of pregnancy,it's necessary to find suitable biomarkers for the early diagnosis and take therapeutic measures accordingly.DNA epigenetic alterations without any changes of the primary sequences have been identified to be correlated with various diseases.DNA methylation alterations are heritable and affected by environmental factors,and it could affect the human susceptibility to various diseases.DNA methylation is a kind of epigenetic mechanism,abnormal DNA methylation has been found to be associated with various pathological processes in placental trophoblasts,such as abnormal placental angiogenesis.Variousgenes with altered DNA methylation levels has been found in PE especially early-onset PE(EOPE)placental tissues.In the research of DNA methylation alterations associated with EOPE placentas,epigenetic pathogenesis of this pregnancy disorder could be explored,and possible epigenetic biomarkers could be found,thus providing new thoughts for the early diagnosis.Canonical Wnt signaling pathway is a highly conserved pathway,and plays crucial roles in the regulation of trophoblast function.Studies have demonstrated that abnormal activation of Wnt signaling pathway is associated with the pathogenesis of various cancers.Abnormal expression of Wnt signaling pathway might result in the trophoblast dysfunction,and contribute to the pathological mechanism of PE.Currently,there are rare published dates on the association between Wnt signaling pathway and PE.WNT2 is a canonical Wnt ligand,which functions as a glycoprotein in autocrine or paracrine modes.WNT2 signaling pathway is implicated in extraembryonic development,placental vasculogenesis,chorioallantoic fusion,and so on.Studies have demonstrated significant decreased levels of WNT2 mRNA and WNT2 protein in preeclamptic placental tissues,and hypermethylation of WNT2 gene promoter,speculating downregulation of WNT2 expression by WNT2 gene promoter hypermethylation.Through verification of the methylation and expression levels of WNT2 in EOPE placental tissues,the possible correlation between WNT2 and EOPE pathogenic mechanism could be studied.DKK1 is a kind of secretory protein,which could inhibit canonical Wnt signaling transduction pathway.DKK1 is associated with the onset of cancer,and is downregulated or silenced in various cancers.In addition to cancers,abnormal expression of DKK1 was found to be associated with skeleton system diseases,rheumatism,diabetes mellitus and Alzheimer disease.Studies showed significant increased levels of DKK1 mRNA and DKK1 protein in preeclamptic placental tissues,which could contribute to the pathogenesis of preeclampsia through inhibiting Wnt signaling pathway.The regulation mechanism of abnormal expression of DKK1 in preeclamptic placental tissues remains unclear,the methylation and expression levels of DKK1 and the correlation between them in EOPE placental tissues are unknown.ObjectiveThe purpose of this study was to investigate the DNA methylation and transcription levels of WNT2 and DKK1 genes and the correlation between them in EOPE placental tissues.Studies in this respect might help understand the physiopathologic mechanism of EOPE and provide new thoughts and methods for the early diagnosis and treatment.Materials and methods 1 Study subjects and classification of groupsThe study included three groups: EOPE group(n=25),normotensive preterm birth(PB)(n=25)group displaying no statistical significance of gestational age with EOPE group and term birth(TB)group(n=25).The PB and TB groups were regarded as the control groups,and the EOPE group as the experimental group.2 Methods2.1 Sample collection and storage: The placental tissues were collected within 15 minutes following caesarean section.After the removal of the membranes,several pieces of placental villous samples in the size of 5×5×5mm were obtained from the center part of maternal side 5mm beneath the upper layer of the placenta.The placental villous samples were washed with cold PBS to remove the maternal and fetal bloods.One portion of the samples was kept directly in the freezing tube for DNA extraction,another portion of the samples was kept in the freezing tube with RNAstore for RNA extraction.Samples were snap frozen in the liquid nitrogen for 10 minutes before storage at-80°C until for use.2.2 DNA extraction and pyrosequencing: Genomic DNA was extracted from 60 mg of placental villous tissues.The DNA was bisulfide-converted,transforming the unmethylated cytosine into uracil.After evaluating the sequences in the promoter regions of WNT2 and DKK1 genes for pyrosequencing,the primers for pyrosequencing were designed,and the reverse primers were labeled with biotin at the 5' end.The methylation levels were determined at 7 CpG sites of WNT2 and 5 CpG sites of DKK1 contained in the target sequences.2.3 RNA extraction and quantitative real-Time PCR: RNA was extracted from 80 mg of placental villous tissues kept in RNAstore with the use of TRIzol Reagent.The extracted RNA was inversely transcribed into single stranded complementary DNA(cDNA)with the use of reverse transcription reagent.Quantitative PCR primers were designed of WNT2 and DKK1 genes.Quantitative real-time PCR was performed to determine the relative expression levels of WNT2 and DKK1 mRNA in all the placental tissues.3 Statistical analysisSPSS 19.0 was used for statistical analysis of the dates obtained.Quantitative dates that satisfied normal distribution,? x ± s was used to display the mean value and the degree of variation.For dates that didn't satisfy normal distribution,median(M)was used to display the mean value,and interquartile range(P75-P25)was used to display the degree of variation.One-Way ANOVA or Kruskal-Wallis test was performed to compare among the three groups,Bonferroni method was further performed to compare between two groups.Pearson or Spearman correlation analysis was performed to analyze the relationship between two groups of variables.P<0.05 was considered statistically significant of the comparision or correlation analysis.Results 1 Clinical parameters of study subjectsThe gestational ages at delivery in the EOPE,PB and TB groups were 33.91±1.39 w,34.14±1.26 w and 38.48±0.88 w,respectively.In comparision,the gestational ages at delivery in the TB group were significantly higher than that in the EOPE group(P<0.05)and the PB group(P<0.05),and there was no significant difference of gestational ages at delivery between the EOPE group and the PB group.The urine protein quantity in the EOPE,PB and TB groups were 3.866±0.823g/24 h,0.235±0.156 g/24 h and 0.235±0.120 g/24 h,respectively.After comparision,the urine protein quantity in the EOPE group was significantly higher than that in the PB group(P<0.05)and the TB group(P<0.05),and there was no significant difference between the TB group and the PB group.The blood pressures(systolic/diastolic)in the EOPE,PB and TB groups were 167.20±18.22/104.76±7.58 mmHg,116.88±7.67/72.13±8.72 mmHg and 111.88±9.24/69.80±9.03 mmHg,respectively.Theblood pressure in the EOPE group was significantly higher when compared with the PB group(P<0.05)and the TB group(P<0.05),and there was no significant difference between the PB group and the TB group.The neonatal birth weight in the EOPE,PB and TB groups were 1740.00±297.55 g,2146.88±277.77 g and 3043.20±921.04 g,respectively.The TB group had a significantly higher neonatal birth weight than the PB group(P<0.05)and the EOPE group(P<0.05),and the neonatal birth weight in PB group was significantly higher than that in the EOPE group(P<0.05).The maternal ages in the EOPE,PB and TB groups were 29.92±6.24 years,31.06±5.25 years and 29.52±4.30 years,respectively.After comparision,there was no significant difference for the maternal ages among the three groups.The fetal gendar(female/male)in the EOPE,PB and TB groups were 15/10,12/13 and 11/14,respectively.After comparision,there was no significant difference for the fetal gendar among the three groups.2 Methylation levels of WNT2 and DKK1 genes in placental tissues of the three groupsThe methylation levels of CpG1,CpG2,CpG3,CpG4 and the average of DKK1 were all significantly higher in the PB group than that in the EOPE and TB groups(P<0.05),and there were no significant differences between the EOPE group and the TB group.CpG5 of DKK1 in EOPE placental tissues was all unmethylated,and the methylation level of CpG5 in EOPE was significantly lower than that in the PB and TB groups,and there was no significant difference between the PB and the TB groups.There were no significant differences for the methylation levels of the 7 CpG sites and the average of WNT2 gene among these three groups(P>0.05).3 Relative mRNA expression levels for WNT2 and DKK1 genes in placental tissues of the three groupsThe relative levels of WNT2 mRNA in the EOPE,TB and PB groups were 1.25±0.77,4.61±1.87 and 4.64±1.68,respectively.The WNT2 mRNA levels in the EOPE group were significantly lower when compared with the PB group(P<0.05)and the TB group(P<0.05),and there was no significant difference between the PB and TB groups.The relative levels of DKK1 mRNA in the EOPE,TB and PB groups were 3.46±1.34,4.01±1.85 and 1.14±0.60,respectively.The DKK1 mRNA levelsweresignificantly lower in the PB placenta tissues than that in the TB placental tissues(P<0.05)and the EOPE placental tissues(P<0.05),and there was no significant difference between the EOPE and the TB placental tissues.In the PB and the EOPE groups,the Pearson correlation analysis showed that the expression levels of WNT2 mRNA were significantly negatively correlated with the expression levels of DKK1 mRNA(r=-0.539,P<0.05).4 Correlation analysis between the methylation levels and transcription levels of DKK1 and WNT2 genesSignificant correlations were not found between the methylation levels of any CpG sites of WNT2 and the levels of WNT2 mRNA.In the PB,TB and EOPE groups,the Pearson correlation analysis showed that the levels of DKK1 mRNA were significantly negatively correlated with the methylation levels of CpG2(r=-0.660,P<0.05)and CpG4(r=-0.357,P<0.05).The levels of DKK1 mRNA were not significantly correlated with the methylation levels of other CpG sites and the average.ConclusionDecreased methylation levels of DKK1 promoter in early-onset preeclamptic placenta tissues may up-regulate the expression of DKK1.The increased expression of DKK1 and decreased expression of WNT2 may be involved in the pathogenesis of early-onset preeclampsia.