Differential Methylation Gene Screening In Preeclampsia Placenta And Expression Of TNFAIP8 In Preeclampsia Placenta And Blood

Epidemiological investigation shows that the incidence of preeclampsia(preeclampsia,PE)was 5%?10%,and shows a rising trend year by year.Its main clinical manifestation includes high blood pressure and proteinuria after 20 weeks of gestation,serious when can cause epilepsy,cerebrovascular accident,retinal detachment,acute liver and kidney dysfunction,disseminated intravascular coagulation,multiple organ failure and so on.It also can cause neonatal premature delivery,intrauterine growth restriction,fetal distress,nerve damage related to anoxia and perinatal deaths.It is one of the leading causes for maternal morbidity and perinatal mortality.Preeclampsia affects not only the pregnancy outcome but also the increasing incidence of motherand childreninvolving chronic diseases subsequently,especially cardiovascular accident,diabetes,heart disease and metabolic syndrome.Development of preeclampsia may involve many factors such as matrix,the placenta and fetal,including trophocyte invasion,immune regulating function,vascular endothelial cell injury,genetic and nutritional factors,etc.But there is still not a single reason can explain all the causes and mechanisms of preeclampsia.Emerging epigenetics,especially DNA methylation may involve more in the physiopathologic mechanism of the disease and has huge potential for research.This study will focus on the methylation profile of placenta with human genome-wide microarray,verify the differentialmethylation candidate genes,explore the possible ways on the pathogenesis of preeclampsiaaccording to its expression in placenta and peripheral blood,and then provide reasonable selection and theoretical basis for peripheral blood prediction and monitoring markers.Chapter 1 Methylation Profile of Preeclampsia Placenta and Biological Function Analysis of Differential Methylation Candidate GenesOBJECTIVEIn our case-control study,we detect DNA methylation level of placenta of early-onset(EOPE),late-onset preeclampsia(LOPE)and control group through the Illumina human methylation450kbeadchip.We analyze the biological function and signaling pathways that the significantly differentmethylation gene loci may be involved according to the specific conditions of screening.Several genetic loci that may be associated with the maternal-fetal interface are focused on and ready for further subsequent validation in order to look for candidate genes related to preeclampsia development.METHODS1.Experimental protocol:Three cases of early-onset preeclampsia,late-onset preeclampsia and control group respectively were included in this research which matched in terms of age,delivery time and gestational age.Their placentas delivered were detected with Illumina human methylation 450k beadchip.2.Placental collection:After delivery the placenta was collected within 30 minutes.The four samples about 1 cm3 size were cut into strips,washed by sterile PBSsolusion,dry,preserved in freezing tube and stored in-80 ? refrigerator.3.Genome DNA extraction and quality control:The placenta DNA was extracted by using DNeasy Blood&Tissue Kit.The quality control was carried on to make the sample concentration not to be less than 55ng/p.l and total amount not to be less than 5 ?g.4.DNA methylation chip:The placenta DNA was dealed with bisulphite conversion,chip hybridization,elution,extension and imaging.The probe hybridization and bisulphite conversion efficiency of the methylation chip were evaluated.The raw data were analyzed using the Illumina GenomeStudio software.5.Biological function clustering analysis of differential methylation genes:We used Pubmed or GALAXY database to identify the position of differential methylation loci in the genome;used MEME database to identify the base sequence combined with CpG island methylation;used DAVID database to analyze the biological function clustering of the diffential methylation genes;used Fischer's exact test to compare the relative importance of each signal pathway after clustering analysis.6.Annotation ofdifferential methylation genes associated with immune:We annotated the gene sequence,description,type,location of chromosome and main function by Pubmed,Genecard and BioGPS.RESULTS1.The result of methylation chip quality control showed that the probe conform to the quality control standard,the experimental system is stable.The fluorescence signal was strong and uniform and the sulfite conversion efficiency is high.2.The methylation chip tested 485,577 loci in the placenta tissue.The total methylation degree of placenta was 0.470 in EOPE group,0.456 in LOPE group and 0.459 in control group respectively.There were no significantlystatistical defferences among them(P<0.05).3.Compared with late-onset preeclampsia and control group,early-onset preeclampsia group found 23308 and 18318 differential methylation genes respectively in the placenta.There were 999 and 527 genes respectively whose difference was statistically significant and their probes were in the TSS or 5 'UTR region.There were about 95 genes in the intersection.4.The differential methylation genesbetween preeclampsiaand normal placenta were mainly associated with the following biological function such as multicellular organismal process,developmental process,multicellular organismal developmental process,anatomical structure development and system development.5.There were 17 and 6 gene loci respectively associated with immunological maternal-fetal interfacein E-C group and L-C group.There were only 2 genes in the intersection which were HLA-H and KIR2DL4.CONCLUSIONWe have successfully tested the the placenta tissue of preeclampsia and normal childbirth by using the global-wide human genome DNA methylation chip.The results shows that there are some methylation level of certain gene loci changed.With the biological information analysis for the chip data,the significantly differentialmethylation genes are involved in multiple information pathways such as cell biological processes and the development of anatomical structures and systems.Due to the large number of genes involved,we can choose some certain genesfor further verification in order to explore the pathophysiological rule and effective methods for diagnosis and treatment of preeclampsia.Chapter 2 Differential methylation candidate genes associated with immune and verification by pyrosequencingOBJECTIVEThrough the preliminary screening of methylation profiles between preeclampsia and normal placenta and classification related to biological function,we focus on the target genesto determine its methylation degree by sequencing.The methylation difference of loci between early-onset preeclampsia,late-onset preeclampsia and normal placenta were verified with pyrosequencing technology.The results would providemore evidence for subsequent protein expression of the gene loci and pathogenesis associated with preeclampsia.METHODS1.Experimental protocol:Totally 46 cases of pregnant women in obstetrics of Fujian provincial hospital from March 2013 to May 2014 were enrolled in the research,which were devided into three groups.There were 16 cases in early-onset preeclampsia group,12 cases in late-onset preeclampsia group and 18 cases in control group.2.Determining target genes for pyrosequencing:The target genes were chosen to satisfy both of the following criteria:their methylation differences had statistical significance(P<0.05,M>1.0 or M<1.0)and the probe distribute at TSS or 5'UTR region.At the same time,theyshould be protein coding genes which were expressed both in placenta and peripheral blood,and their basic methylation level werehigh.3.Genome DNA extraction and quality control:The placenta DNA was extracted by using DNeasy Blood&Tissue Kit.We use agarose gel electrophoresis to ensure DNA concentration was more than 2?g.4.Pyrosequencing:Primers were designedfirst.About 1?2?g sample was taken to convert by EpiTect Plus DNA Bisulfite Kit.The DNA samples transformed as templating were carried on PCR.PCR products were tested by PyroMark Q24 real-time quantitative pyrophosphate analyzer.RESULTS1.Determining target genes:excluded pseudogenes,select one of the genes in the intersection,KIR2DL4.TNFAIP8 is one of significantly different genes chosen in early-onset preeclampsia group compared with control group,because TNFAIP8 expression level is higher in placenta and lymphocytes of peripheral blood.It was closely related to maintaining cell stability and regulating immune function.2.The age,pregnant or delivery time had no significantly statistical difference among three groups.The placenta DNA quality evaluation was good.5?The methylation levelof KIR2DL4two loci were higher,about 50%and 70%respectively.But the differences among the three groups had no significantly statistical significance(P>0.05).3.The methylation level of TNFAIP8 two loci was approximately 10%.At a TNFAIP8 locus 1,the methylation level of early-onset preeclampsia group compared with the other two groups were significantly different(P<0.03).And late-onset preeclampsia group compared with control group had no significantly statistical difference(P>0.05).And the methylation level of TNFAIP8 locus 2had no significantly statistical difference among three groups(P>0.05).CONCLUSIONThe loci chosen to verify must consider the organs of the gene expression and possible ways to participate in the preeclampsia development in addition to gene type,basic level of methylation.Otherwise it will increase the risk of sequencing validation and waste economic resources.Early-onset preeclampsia compared with late-onset preeclampsia and normal women has significantly decreased methylation level of TNFAIP8.But there were no significant differences in KIR2DL4 gene.The next step we will expand to location and quantity of TNFAIP8 expression in the placentain order to deepen the research from the gene level to molecular level.Chapter 3 Difference of TNFAIP8 expression between preeclampsia and normal placentaOBJECTIVEAt present TNFAIP8 expression in placenta and its role in the pathogenesis of preeclampsia has not yet being reported.lt is critical to study TNFAIP8 expression way for trophocyte invasion andrebuildig vessels.This study will explore the location and quantityof TNFAIP8 expression in placenta and the relationship associated with the methylation condition of the genesdetected by the chip previously,which will help to clarify the mechanisms of preeclampsia pathological damage.METHODS1.Experimental protocol:Totally 64 cases of pregnant women in obstetrics of Fujian provincial hospital from March 2013 to August 2014 were enrolled in the research,which were devided into three groups.There were 23 cases in early-onset preeclampsia group,16 cases in late-onset preeclampsia group and 25 cases in control group.2.Placental collection:After delivery the placenta was collected within 30 minutes.The four samples about 1 cm3 size were cut into strips,washed by sterile PBS solusion,dry,preserved in freezing tube and stored in-80 ? refrigerator.3.Placenta immunohistochemicalstaining:About 0.5cm X 0.5cm X 0.1cm size of fresh placenta was washed by PBS and fixed with 4%paraformaldehyde.Paraffin embedding tissue block was sliced,dewaxed and stained by immunohistochemistry.In the experimental group and the control group,the positive and negative images were chosen to take 100 X or 400 X micrography and analyzed.4.Placental fluorescence quantitative RT-PCR:The gene primers were designed for the target genes.The total RNA was extractedby RNA extraction kit.The agarose gel electrophoresis was used to detect the integrity of the RNA.ResidualDNA in the genome RNA was digested.After reverse transcription the product was put on fluorescence quantatitive PCR instrument.The data were analyzed for relative quantity.5.Placental Western Blot:we extracted the total protein of placenta and test the protein content of the samples.After SDS-PAGE gel preparation,protein denaturation and electrophoresis,protein bandsseparated from the gel was transfered to the solid phase by electrophoresis.Then the sample was incubated with the antibody,tested,washed,exposed and developed a film.At last its grey value was analyzedby image J software.RESULTS1.Placenta immunohistochemical results:TNFAIP8 expressed positive in the syncytiotrophoblast and vascular endothelial cells of placental villi structure.Comparedlate-onset preeclampsia and normal placenta,early-onset preeclampsia placenta had significantlyincreasing positive cells,darker staining and higher IHS scores.2.Placenta fluorescence quantitative RT-PCR results:Because the standard curve had high linear photometric phase and amplification efficiency,this method could be used to amplifythe sample for analysis.Amplification curve and dissolution curve showedthat target gene and actin gene had efficient amplification and single product.TNFAIP8mRNA relative expression in placenta had significantly statistical difference among three groups(P=0.000).TNFAIP8mRNA level of early-onset preeclampsia was obviously higher than that of late-onset preeclampsia and the control group(P=0.000).And TNFAIP8 mRNA levelof late-onset preeclampsia was obviously higher than that of control group(P=0.000).3.Placental Western Blot results:The electrophoresis stripesof TNFAIP8 decreased according to the sequence of early-onset preeclampsia,late-onset preeclampsia and normal placenta.TNFAIP8 protein level in placenta had significantly statistical difference among three groups(P=0.000).TNFAIP8 protein level of early-onset preeclampsia was obviously higher than that of late-onset preeclampsia and the control group(P=0.000).And TNFAIP8 protein level of late-onset preeclampsia was obviously higher than that of control group(P=0.000).CONCLUSIONTNFAIP8 gene has significantly low methylation according to the previous results of placental chip and pyrosequencing.TNFAIP8 transcription and expression should be increased obviously by general rules.This chapter have validated the speculation.TNFAIP8 expression in placenta of early-onset preeclampsia is obviously higher than that of late-onset preeclampsia and normal pregnant women,and TNFAIP8 expression of late-onset preeclampsia is significantly higher than that of normal placenta.Therefore we speculate that TNFAIP8 gene in placenta of early-onset preeclampsia and late-onset preeclampsia havethe similar pattern of methylation profile and protein expression.Chapter 4 Difference of TNFAIP8 expression between preeclampsia and normal peripheral bloodOBJECTIVEThe previous study had found that TNFAIP8 gene had significantly lower methylation levels in placenta and high levels of transcription and expression,whichwas one of the important influent factors might be involved in preeclampsiaetiology.There had been some literaturesthat confirmed TNFAIP8 expression in lymphoid tissue,but only reported in the thymus,bone marrow and so on.Whether TNFAIP8 expressed in blood lymphocytes,and its expression reflected the placental pathological change was not studied.This chapter would explore the feasibility of TNFAIP8 as markers though the TNFAIP8expression rule in blood.METHODS1.Experimental protocol:Totally 64 cases of pregnant women in obstetrics of Fujian provincial hospital from March 2013 to August 2014 were enrolled in the research,which were devided into three groups.There were 23 cases in early-onset preeclampsia group,16 cases in late-onset preeclampsia group and 25 cases in control group.2.Blood collection:The 3ml×2 peripheral bloodwas drawn fromelbow vein of preeclampsia and normal pregnant women.The blood was put in EDTA tube and stored in-80 ? refrigerator.3.Blood fluorescence quantitative RT-PCR results:The experimental steps processed as the third chapter.4.Western Blot results of Blood lymphocytes:Total protein was extracted from blood lymphocytes.The pyrolysis liquid was added into the cells to lyse cells.The cultured cells were collected to centrifuge for 3-5 minutes.We took supernatant and continued the experiment.The experimental steps remaining were same to the third chapter.RESULTS1.Blood fluorescence quantitative RT-PCR results:Real-time amplification curve and dissolution curve showed that target gene and actin gene had efficient amplification and single product.The relative expression of TNFAIP8mRNA inblood lymphocyteshad significantly statisticaldifference among three groups(P=0.000),TNFAIP8mRNA level of early-onset preeclampsia was obviously higher than that of late-onset preeclampsia group and the control group(P=0.000).TNFAIP8mRNA level of late-onset preeclampsia was obviously higher than that of control group(P=0.000).2.Blood lymphocyte Western Blot results:The electrophoresis stripes of TNFAIP8 protein decreased according to the sequence of early-onset preeclampsia,late-onset preeclampsia and control group.TNFAIP8 protein expression in blood had significantly statistical difference amongthree groups(P=0.000).TNFAIP8 protein level of early-onset preeclampsia was obviously higher than that of late-onset preeclampsia and the control group(P=0.000).And TNFAIP8 protein level of late-onset preeclampsia was obviously higher than that of control group(P=0.000).CONCLUSIONTNFAIP8expression in blood lymphocytesis similar to that in placenta.TNFAIP8 transcription and protein expression decreased in the order of early-onset preeclampsia group,late-onset preeclampsia group and control group.And TNFAIP8 expression is significantly different among three groups.The further study of blood is expected to provide the theory basis for early clinical prediction and judging the severity of preeclampsia.SUMMARY1.The human genome DNA methylation chip(Illumina HumanMethylation450 BeadChip)is successfullyapplied to analyze the methylation profile in placenta of early-onset preeclampsia,late-onset preeclampsia and normal delivery.2.The overall average methylation levelin placenta of early-onset preeclampsia,late-onset preeclampsia andcontrol grouphave no significantly statistical difference.3.Compared with the control group,differentially expressed genes in early-onset preeclampsia and late-onset preeclampsia exist in the placenta tissue.And these genes are involved in cell growth and proliferation,mainly including the biological features such as multicellular organismal process,developmental process,multicellular organismal developmental process,anatomical structure development and system development.4.The target genes for validation are selectedfrom the methylation chip screening according to the genetic types,basic level of methylation,the organ for research and the ways to participate in the development of preeclampsia.Otherwise it will increase the risk of sequencing validation and waste economic resources.5.Placental TNFAIP8 methylation significantly decreases or even does not happen inearly-onset group compared with late-onset and normal group.TNFAIP8 mainlylocates in syncytiotrophoblast cells and vascular endothelial cells of placenta villi which may be related to trophocyteinvasion and vascular endothelial cell function.The transcription and translation of TNFAIP8 in preeclampsia placenta is higher than normal placenta,and early-onset level is significantly higher than late-onset level.So we speculate that it may participate in the development of preeclampsia.6.TNFAIP8 has low level expression in normal placenta which may be related to maintain the steady state of normal immune function.7.The difference of two KIR2DL4 loci verified by pyrosequencinghas no statistical significance.Theseloci may participate in the pathogenesis of preeclampsia through non-methylation mechanism or need to increase the number of samples for further research.8.Compared with normal pregnant women,preeclampsia women have significantlyhigherexpression level of TNFAIP8 in blood lymphocytes.And early-onset group has more expression level than late-onset group,which is expected to be as the early prediction of preeclampsia and the markerreflecting disease progression.