Role Of Polymorphism In Cyclophilin A Gene And Its Effects In The Development Of Severe Preeclampsia

BackgroundPreeclampsia(PE)is a unique,severe obstetric complication during pregnancy.The main clinical manifestations of PE were severe high blood pressure,proteinuria,and complications such as renal and heart insufficiency,liver involvement,fetal intrauterine growth restriction(IUGR),and even fetal intrauterine death.PE is a main cause of maternal and fetal mortality and morbidity,which seriously threat the maternal and child health.Most patients would gradually relieve after termination of pregnancy,but there are still some patients with irreversible organ damage such as liver,kidney and brain.According to the gestational weeks of onset,severe PE can be divided into early-onset PE(<34 weeks)and late-onset PE(? 34 weeks).Early-onset severe PE is more serious,progressing faster,more likely to cause functional damage,higher incidence of maternal and infant adverse outcomes,and more difficult in clinical treatment.The key consequences are uterine spiral artery recast disorder and systemic vascular endothelial system injury.At present,the pathogenesis of PE remains elusive.Therefore,it is very important to elucidate the damage mechanism of trophoblast cells in early pregnancy for clarifying the pathogenesis of PE and finding the target of clinical drug therapy.Inflammatory immune overactivation is one of the important mechanisms of PE.It has been found that the inflammatory reactions in maternal-fetal interface and systemic endothelial system were over-activated in severe PE patients,and the expression levels of TNF-a,IL-6 and IL-1? in peripheral blood and placenta tissues of PE patients were significantly increased.At present,the molecular mechanism of the over-activation of inflammatory factors is still unclear.Any factors that increase the expressions of inflammatory factors may be involved in the pathogenesis of PE.Soluble fms-like tyrosine kinase-1(sFlt-1)is an important anti-angiogenesis factor.It can competently bind to VEGF and PLGF,activating oxidative stress,leading to systemic vascular endothelial injury and elevating blood pressure,which could induce PE.Hypoxia inducible factor-la(HIFla)is an important transcription factor,which can negatively regulate the expression of vascular endothelial growth factor and inhibit angiogenesis.It was found that the expressions of sFlt-1 and HIF1? in placentas of severe PE patients were significantly increased.Therefore,sFlt-1 and HIF1? are important molecules involved in the pathogenesis of severe PE.Cyclophilin A(CypA)is the most widely distributed and abundant pro-inflammatory factor in the Cyclophilin family.CypA has intracellular and extracellular forms.Intracellular CypA(iCypA)is involved in protein folding,transporting and function,regulate cellular functions,T cell subtype differentiation,platelet activation and cytokinesis.The extracellular CypA(eCypA)is secreted by autocrine or paracrine pathways under hypoxia,infection,and oxidative stress,mediating intercellular communication and information transmission,and involved in cell apoptosis,invasion and migration,and oxidative stress.It has been proved playing important roles in the development of cardiovascular diseases,such as atherosclerosis,coronary heart disease,pulmonary hypertension,and so on.Since many common features and pathological changes between cardiovascular diseases and severe PE,eCypA may also be involved in the pathogenesis of severe PE.The coding gene of CypA is Peptidylprolyl isomerase A(PPIA),located in 7p13 chromosome region and containing multiple genotypes.Studies have shown that CypA polymorphism is closely related to the incidence and severity of coronary heart disease.To date,there are no reports on the correlation between CypA gene polymorphism and preeclampsia world widely,and the role and mechanism of CypA in the pathogenesis of severe preeclampsia is still unclear.In this study,we investigated the correlation between CypA polymorphism and severe preeclampsia,detected the CypA expression in maternal serum and placentas,and furtherly discussed the influences of CypA on JEG3 cell to investigate its possible molecular mechanism in vitro experiment,to provide possible theoretical support and experimental basis for the pathogenesis and clinical treatment of preeclampsia.Part ?Association between polymorphism in Cyclophilin A gene with severe preeclampsiaObjectiveTo investigate the association between polymorphism in CypA gene and severe preeclampsia.MethodsA total of 261 pregnant women were recruited from the Department of Obstetrics in our hospital between July 2015 and September 2017,including 82 patients diagnosed with severe preeclampsia in PE group and 179 healthy age-matched pregnant women in control group.PE group contained 38 patients with early-onset PE and 44 patients with late-onset PE.The polymorphism disequilibrium of PPIA was analyzed by Haploview 4.2 software,and three tagging SNP sites(rs3735481,rs9638978 and rs 11984372)were chosen for analysis using the Tagger analysis method.SNPs genotyping was conducted by TaqMan assays.The allele frequency and the association between different genotypes and severe preeclampsia in codominant,dominant,recessive and overdominant modes were analyzed,and the correlation between polymorphism in CypA gene and early/late onset PE was further analyzed.Results1.There were AA,AC and CC three allele genotype of SNP rs3735481.The CC genotype was the least,AA genotype was the most.The frequency of allele C in PE group(14.6%)was significantly lower than the control group(22.3%)(OR:0.6,95%CI:0.36-0.98;P<0.05),suggesting that allele C may play a protective role in the pathogenetic process of severe PE.2.There were AA,AG and GG three allele genotype of SNP rs9638978.There was no significant difference in genotype composition ratio between the two groups(P>0.05).The frequency of allele A in PE group(37.20%)was significantly higher than that in control group(27.65%)(OR:1.55,95%Cl:1.05-2.29,P<0.05),suggesting that allele A may play a pathogenic role in the pathogenetic process of severe PE.3.There were only AA and AC allele genotype of SNP rsl 1984372.The proportion of AA genotype and allele A was obviously higher in two groups(92.68%vs.94.41%,96.34%vs.97.21%).There was no significant difference in allele type composition ratio and allele frequency between the two groups(P>0.05).4.We evaluated genotype distribution of rs3735481 and rs9638978 in four genetic models.There was no significant difference in the four genetic models between the PE group and control group(P>0.05).Because there was no CC genotype in SNP rs11984372,we only made analysis in co-dominant genetic model and showed no statistical difference between the two groups(P>0.05).5.The PE group was further divided into two groups:early-onset severe PE group(e-PE group,n=38)and late-onset severe PE group(1-PE group,n=44).Compared with the control group,there was no significant difference in allele frequency and genetic model analysis of SNP rs3735481 and rsl 1984372 in e-PE group and 1-PE group(P>0.05).The frequency of SNP rs9638978 allele A in the e-PE group was significantly higher than that in the control group(P=0.002).For SNP rs9638978,significant differences were found in the co-dominant model AA vs.GG(OR:5.75,95%CI:2.01-16.48,P<0.001),the dominant model AA+AG vs.GG(OR:2.29,95%Cl:1.09-4.82,P<0.05),and the recessive model AA vs.GG+AG(OR:4.32,95%Cl:1.67-11.17,P<0.01).while there was no significant difference in allele frequency and four genetic models of rs9638978 between 1-PE group and control group(P>0.05).Conclusion1.The CypA genetic polymorphisms of rs3735481 and rs9638978 may be associated with severe PE.2.Allele C of CypA rs3735481 may play a protective role while allele A of CypA rs9638978 may play a pathogenic role in the pathogenesis of severe preeclampsia.3.CypA rs9638978 AA genotype may be associated with an increasing risk of early onset severe PE,and may be one of its susceptible genes.Part ?Association between Cyclophilin A and severe preeclampsiaObjectiveTo investigate the relationship between CypA expression in peripheral blood and placental tissues and severe preeclampsia(PE),and further explore the relationship between CypA gene expression and severe PE.MethodsPregnant women were chosen from the first part of the research according to the age matching principle.The peripheral blood and placental tissues were collected.The expression of CypA in peripheral blood plasma was detected by ELISA,the CypA mRNA level in placental tissues was detected by qRT-PCR,and the expression of CypA protein in placental tissues was detected by Western blotting,immunohistochemistry and immunofluorescence methods.Results1.The levels of CypA in peripheral blood was significantly increased in PE group compared with control group(P=0.0003)by ELISA method.Further analysis shows that the CypA levels of e-PE group and 1-PE group in peripheral blood were significantly higher than that of control group(P<0.01),and highest in the e-PE group,but no statistically significant difference was found between e-PE group and 1-PE group(P>0.05).2.Pearson correlation analysis showed that the CypA levels in peripheral blood was correlated with maternal systolic blood pressure and diastolic blood pressure(P<0.05).3.CypA serum levels were significantly lower in carriers with the AC genotype than that in carriers with the AC genotype of rs3735481(P<0.05),and significantly higher in carriers with the AA genotype than that in carriers with AG and GG of rs9638978(P<0.05).No significant difference was found between carriers with AG and GG of SNP rs9638978 and between carriers with AA and AC of SNP rs11984372(P>0.05).4.The levels of CypA mRNA and protein expression in placental tissues of PE group were significantly higher than that of control group by qRT-PCR method(P<0.05).Further analysis showed that the CypA mRNA levels in the placental tissues of e-PE group and 1-PE group were significantly higher than that of control group(P<0.05),and highest in the placental tissues of e-PE group.5.Western blotting showed that CypA protein was also expressed in normal placental tissues,and protein level was significantly higher in placental tissues of patients in PE group than that in control group(P<0.05).Immunofluorescence and immunohistochemistry tests further proved that CypA protein was highly expressed in placental tissues of PE group and mainly located in the extracellular matrix.Conclusion1.The CypA expression in peripheral blood were significantly increased in patients with severe PE,especially of patients with early-onset severe PE,and the serum levels of CypA were correlated with systolic and diastolic blood pressure.These results suggest that high expression of CypA in peripheral blood may be involved in the pathogenesis of severe PE.2.The CypA expression in placenta was significantly increased in patients with severe PE,which suggesting that the high expression of CypA in placenta may be involved in the pathogenesis of severe PE.3.The differential expression of CypA in peripheral blood is associated with gene polymorphism.SNP rs3735481 allele C may play a protective role while rs9638978 allele A may play a protective role in the onset of severe PE.Part ?Mechanism of excellular cyclophilin A in the pathogenesis of preeclampsiaObjectiveTo investigate the effect of exocellular Cyclophilin A(eCypA)on the inflammatory factors and apoptosis of JEG3 cells,as well as the expression of sFLT1,and to further investigate the effects of eCypA on HIF1? signaling pathway by silencing HIF1? through small interfering siRNA,so as to clarify the possible mechanism of eCypA involved in the pathogenesis of preeclampsia.MethodsTo cultivate human trophoblasts JEG3 cells in vitro.Exogenous human recombinant CypA(hrCypA)protein was used to intervene cells.The mRNA expression of inflammatory factors(TNF-a,IL-6 and IL-1?)was detected by qRT-PCR.The influence of hrCypA on JEG3 apoptosis was detected by flow cytometry and TUNAL assay.Western blotting was used to detect the expressions of p-65/p-p65,I?Ba/p-I?B?,caspase3/cleaved caspase3,parp1/cleaved parp1,P53,FLT-1 and HIF-la.ELISA was used to detect the effects of hrCypA on secretions of sFlt-1,IL-1? in JEG3 cells.Meantime,JEG3 cells was transfected by small interfering RNA to inhibit the expression of HIF1?,then the expressions of inflammatory cytokines IL-1?,p-65/p-p65,FLT-1,sFLT-1,and cell apoptosis were further detected.Results1.qRT-PCR showed that the mRNA levels of TNF-a,IL-6 and IL-1? were increased significantly after hrCypA intervention in JEG3 cells(P<0.05),and increasing with the increase of hrCypA concentration.Western blotting showed that hrCypA significantly increased the expression of p-p65 and p-I?B? in JEG3 cells(P<0.05).With increase of hrCypA concentration,the expression of p-p65 and p-I?B? showed an increasing trend.2.Flow cytometry and TUNEL assay showed that hrCypA significantly promoted the apoptosis of JEG3 cells(P<0.05),and the apoptotic rate was increasing with the increase of hrCypA concentration.Western blotting showed hrCypA significantly increased the expression of cleaved caspase 3 and cleaved parpl in JEG3 cells(P<0.05).The expression of P53 was also increased,but the difference was not statistically significant(P>0.05).3.qRT-PCR showed that the FLT1 mRNA level was significantly increased in hrCypA stimulated JEG3 cells(P<0.05),and the level was increasing with the increase of hrCypA concentration.Furtherly,Western blotting analysis showed that expression of FLT1 was significantly increased(P<0.05).ELISA showed that sFltl secretion was also significantly increased(P<0.05),and level was increasing with the increase of hrCypA concentration.4.Western blotting showed that hrCypA significantly increased the expression of Hif-1? protein in JEG3 cells(P<0.05).After Hif1? siRNA was transfected into JEG3 cells,qRT-PCR showed that the expression of Hifla mRNA was significantly inhibited(P<0.05).5.Then hrCypA was used to intervene the infected cells.Western blotting showed that levels of FLT1 and p-p65 were significantly increased in CypA/NC group compared with the NC group,while significantly inhibited in CypA/siHifla group(P<0.05).ELISA detection showed that the levels of sFLTl and IL-1(3 were also obviously increased in CypA/NC group than the NC group,while significantly inhibited in CypA/siHifla group(P<0.05).TUNEL detection showed that the cell apoptosis increased significantly in the CypA/NC group compared to the NC group,while significantly reduced in the CypA/siHifla group,and the staining degree of apoptotic cells was significantly reduced.Conclusion1.eCypA can promote inflammation of JEG3 cells.2.eCypA can promote apoptosis of JEG3 cells by caspase 3 pathway.3.eCypA can up-regulate the secretion of sFlt1 in JEG3 cells.4.eCypA can activate Hifla signaling pathway in JEG3 cells.After silenced by Hif1?-siRNA,the expression of IL-1??p65?sFLT1 and the apoptosis of JEG3 cells induced by eCypA were significantly suppressed.It is suggested that eCypA may mediate its pro-inflammatory,pro-apoptotic and sFLT1 secretion effects on trophoblasts through Hif1? signaling pathway.