DNA Methylome In Placental Tissues Of Preeclampsia,Preterm And Term Pregnancy

Preeclampsia(PE)is a gestational hypertension disorder with hypertension,proteinuria and other systemic disorders occur after 20 weeks of pregnancy.Preeclampsia is one of the leading causes of increased maternal and infant mortality,as well as low birth weight,premature delivery and stillbirth.There have been a lot of studies on preeclampsia by scholars at home and abroad,but the pathogenesis of preeclampsia is complicated and has not been fully clarified.Placenta is an important organ in direct contact between mother and fetus.As the community of mother and fetus,placenta is crucial to the growth and development of fetus in utero and the outcome of pregnancy.The rapid disappearance of clinical symptoms of preeclampsia after delivery or placentectomy suggests that placental factors play an important role in the pathogenesis of preeclampsia.In recent years,a large number of studies have found that the expression profile of preeclampsia placenta tissues is significantly changed compared with the normal control group,suggesting that epigenetics plays an important role in the development of preeclampsia.However,the genomic methylation level of placenta tissues is affected by a variety of factors.Some studies have found that placentas at different gestational ages have different methylation profile.ObjectiveIn this study,Infinium HumanMethylation 850K Beadchip platform was used to analyze genome-wide methylation profile of preeclampsia,preterm and term pregnancy placental tissues.After data analysis,the differential methylation genes that may be related to preeclampsia were screened for verification,and signal pathways related to functional enrichment analysis,and the changes in the methylation levels of these genes were discussed whether these genes were related to the pathogenesis of preeclampsia.And to provide a new basis for the pathogenesis of preeclampsia.Materials and methods1 Study subjectsThe maternal placental tissues of the preeclampsia(PE)group,preterm birth(PB)group and term birth(TB)pregnancy were selected,with 24 cases each.Early onset severe preeclampsia with gestational age<34 weeks was selected as PE group,and preterm delivery(28-37 weeks of gestation)due to non-specific diseases such as premature rupture of membranes,placenta previa,and cervical dysfunction was selected as PB group.The gestational weeks of the two groups were matched.Delivered after 37 weeks of gestation without complications were selected as TB group.2 Methods2.1 Collection of placental tissuesPlacental tissues were collected within 15 minutes after cesarean section(about 5×5×5mm、60mg),near the maternal side under the surface of 5mm.Washed with cold PBS to remove the maternal and fetal blood,and then saved in frozen storage tubes.Avoiding visible necrotic,infarcted or calcified tissues.The placental tissues which prepared for RNA extracted were placed in RNA preservation solution.Put the placental tissues in liquid nitrogen for 10 minutes,and then stored in-80 C°refrigerator for use.2.2 Genomic DNA extraction and Infinium HumanMethylation 850K Beadchip genome-wide detectionGenomic DNA was extracted from 60mg placental tissue.The extracted DNA was examined using NanoDrop 2000 and agarose gel,qualified samples were bisulfide-converted,transforming the unmethylated cytosine into uracil.Transformed samples to do Infinium HumanMethylation 850K Beadchip genome-wide detection.2.3 The data analysis of Infinium HumanMethylation 850K Beadchip2.3.1 Raw data preprocessingAfter filtering the original data(idat file),it is standardized according to the Peak Based Correction(PBC)algorithm.The standardized β-value was used to measure the methylation level of the detected sites,and the to compare the differences between the three groups.2.3.2 The analysis of differential methylated sites(DMSs)The screening of DMSs was performed by calculating the P value of the differential methylated sites using paired t-test in the R,and then using Benjamini&Hochberg for multiple tests to get the adjusted P value.The differential methylated sites were screened by adjusted P value and delta β.2.3.3 The analysis of differential methylated regions(DMRs)Differential methylated sites often appear as clusters in the genome and form a segment of differential methylated regions.DMRs represents the hypomethylated or hypermethylated of a region on chromosome,ranging from a few hundred bp to the Mb level.The screening of DMRs was performed using the ChAMP package and the Probe Lasso method.2.3.4 Functional enrichment analysisFunctional enrichment analysis is to annotate and categorize the GO(Gene Ontology)function and Pathway in KEGG database for whole genome and differential methylated genes.The two methods were used to analyze the functional enrichment of differential methylated sites’ nearest neighbor genes and genes in the differential methylated regions.2.4 PyrosequencingThe regions of differentially methylated genes(CMIP,BLCAP and MICA)were selected for evaluation,and pyrosequencing primers were designed by PyroMark Assay Design 2.0.Biotin was labeled at the 5 ’end of the reverse primers.DNA transformed by disulfite was prepared to verify the methylated level of differentially methylated genes.2.5 The extraction of RNA and quantitative real-time PCRRNA was extracted from placental tissues and then was reversely transcripted into cDNA.The primers were designed and the mRNA expression levels of CMIP,BLCAP and MICA genes were detected by quantitative real-time PCR.3 Statistical analysisSPSS 19.0 and GraphPad Prism 7.0 were used for statistical analysis.The correlation between the two groups of data was analyzed by Student’s-t correlation coefficient,and the significant difference between the groups was analyzed by one-way analysis of variance(ANOVA).β-value which represents the methylated levels of Infinium HumanMethylation 850K Beadchip was tested by non-parametric Mann-Whitney test.α=0.05 was used as the test level.Results1 Clinical features of studied subjectsThe gestational age of PE group and PB group was significantly lower than that of TB group(P<0.01),while there was no significant difference between PE group and PB group(P>0.05).The blood pressure and urine protein in PE group were significantly higher than those in TB group and PB group(P<0.01).The birth weight of PE and PB group was significantly lower than that of TB group(P<0.001),and the birth weight of PE group was significantly lower than that of PB group(P<0.001).There was no significant difference in maternal age and fetal sex among the three groups.2 The DNA methylation level of the three groupsThe global methylation level of placental tissues in PE and PB groups was significantly higher than that in TB group,suggesting that the methylation level of placental tissues may be closely related to gestational ages.Excluding the influence of gestational weeks,there were 808 differential methylated sites in the PE group,among which 524 were hypomethylated and 284 were hypermethylated.There were 137 differential methylated genes,and these differential methylated genes were located on chromosomes and annotated,among which the most significant differences were CMIP,BLCAP and MICA.3 Verification of differential methylated genesThe differential methylated genes(CMIP,BLCAP and MICA)screened out by the 850K chip were verified by pyrosequencing and quantitative real-time PCR.The result showed that CMIP gene and BLCAP gene were hypomethylated in PE,and their mRNA expression levels were both increased.However,the methylation level and mRNA expression level of MICA gene were not significantly different among the three groups.4 Functional enrichment analysisDAVID 6.7 was used to analyze the GO and KEGG pathway enrichment of differential methylated genes.Differential methylated genes were significantly enriched in three functional subclasses:biological function,cellular component and molecular function,which mainly included:cell adhesion,development of nervous system,biological adhesion,systematic development and development of multicellular organs.Among them,232 differential methylated genes are involved in cell adhesion,which is the most significant biological process of enrichment.The significantly enriched signaling pathways include pyruvate metabolism,glyceride metabolism,phospholipase D signaling pathway,PI3k-Akt signaling pathway,cGMP-PKG signaling pathway,and ECM-receptor interaction.Among them,the PI3k-Akt signaling pathway is the signaling pathway with the highest enrichment of differential methylated genes.Other significantly enriched pathways include cAMP,Wnt and MAPK signaling pathways.ConclusionThe methylation level of placental tissue is closely related to gestational ages,and the global methylation level of preeclampsia placental tissue is decreased.The study of DNA methylation in placental tissues may provide support and direction for the study of pathogenesis.