| Part I Association study of ERAP1 single nucleotide locus with genetic amplification validation in pregnant women with pre-eclampsia in ShandongObjective: Our group identified that three single nucleotide polymorphic loci(rs30187,rs27044,and rs469783)at endoplasmic reticulum aminopeptidase 1(ERAP1)is associated with the development of pre-eclampsia by exome resequencing association analysis.In order to confirm whether ERAP1 is a genetic susceptibility factor for pre-eclampsia,we further validated the association of these three single nucleotide sites with the occurrence of pre-eclampsia by genetic amplification in Shandong population.Methods: Based on the pre-eclampsia specimen library established by our group previously,990 pre-eclampsia patients were used as the case group and 1240 healthy pregnant women were used as the control group.We obtained DNA sample from the peripheral blood of the study subjects,and genotypic analysis of these three loci was performed by Taqman probe fluorescent PCR technique.Ten samples randomly selected from each locus were subjected to PCR amplification,and the bases represented by the probe start signal were verified using first-generation sequencing technology.Statistical analysis of the frequency distribution of genotypes and alleles between the case and control groups was performed.Results: 1.We analyzed the clinical data of the case and control groups and found that there were significant differences between the two groups(P<0.05)in indicators such as week of delivery,weight gain during pregnancy,systolic blood pressure,white blood cells,diastolic blood pressure,and triglycerides.However,there was no significant difference in neutrophil counts and the number of miscarriages(P>0.05).2.For the rs30187 locus,the genetic distribution was statistically different between the case and control groups(genotype: c2=29.246,P<0.0001;allele: c2=4.682,P=0.03,OR=1.060,95%CI=1.005-1.118);the rs27044 locus was statistically different between the two groups in genotype distribution(c2=28.949,P<0.0001),while the allele frequency distribution was not significantly different(c 2=2.306,P=0.129,OR=1.054,95%CI=0.985~1.128);the genotype and allele frequency distributions of the rs469783 were significantly different between the two groups(genotype: c2=7.012,P= 0.03;alleles:c2=6.452,P=0.011,OR=1.082,95%CI=1.018~1.150).Genetic analysis of the dominant and recessive gene models at the three loci revealed that rs30187 and rs27044,both recessive models,were statistically different between the two groups and that the CC genotype at both loci increased the risk of developing pre-eclampsia(rs30187: c2=20.817,P<0.0001,OR=1.380,95%CI = 1.200~1.587;rs27044: c2=19.966,P<0.0001,OR=1.518,95%CI=1.261~1.827).For the rs469783 locus,the dominant gene pattern showed a statistical difference between the two groups(c2=5.823,P=0.016,OR=0.836,95%CI=0.723~0.968),and genotype CC is a protective factor for the development of pre-eclampsia.3.Further analysis of the correlation between single nucleotide polymorphism and clinical phenotypes revealed that the genotypes of rs30187 and rs27044 were correlated with diastolic blood pressure(F=6.923,P<0.0001;F=4.460,P=0.004)while the rs469783 was not significantly correlated with clinical phenotypes.Conclusion: ERAP1 is associated with genetic susceptibility to pre-eclampsia,and carriers of the CC genotype at the rs30187 and rs27044 are at higher risk for pre-eclampsia,whereas the CC genotype at the rs469783 is a protective factor for the development of pre-eclampsia.Part II Study on the involvement of ERAP1 missense variants in the pathogenesis of pre-eclampsiaObjective: Endoplasmic reticulum aminopeptidase 1(ERAP1)is able to catalyze the conversion of angiotensin II to angiotensin III and IV in vitro,which is involved in the regulation of blood pressure and may play a role in the pathogenesis of pre-eclampsia.The first part of this study showed that ERAP1 single nucleotide locus is associated with pre-eclampsia.Therefore,in this study,we constructed two missense variants of this gene for cellular and molecular in vitro experiments to preliminarily explore the molecular pathogenesis of ERAP1 in preeclampsia..Methods: Wild-type plasmids were constructed,and point mutation primers were designed to construct two missense variant plasmids using the wild-type plasmids as templates.si RNA-scramble(control group),si RNA-ERAP1,pc DNA3.1+ null plasmid,ERAP1 wild-type plasmid,ERAP1-c.1583T>C variant plasmid,ERAP1-c.2188G>C variant plasmid were transfected into Htr8 cells,and cellular RNA and protein were extracted to detect the expression level of ERAP1.We used transwell migration assay,transwell invasion assay,cell scratching assay and CCK-8 cell proliferation assay to observe changes in cell function.Statistical analysis using two independent samples t-test concluded that P<0.05 was statistically significant.Results: 1.After cells were transfected with si RNA-ERAP1,compared with the control group: the expression of ERAP1 was significantly decreased at both m RNA and protein levels(m RNA: P=0.0017,protein: P=0.0482),indicating that si RNA transfected with the target gene was able to knock down the expression of the gene.2.After knockdown of the target gene: the absorbance value of cells at 450 nm was significantly decreased(P=0.0458),indicating that the cell proliferation ability was inhibited;the cell migration rate was significantly decreased after 48 h of scratching(P=0.0309),and the number of cells crossing transwell chambers at 24 h was significantly decreased(P=0.0302),indicating that the migration ability of cells in the knockdown group was decreased;the number of cells crossing matrix gel-containing transwell chambers after 24 h was significantly reduced(P=0.0464),indicating that the invasive ability of cells was inhibited.3.After transfection of wild-type plasmid into cells,the m RNA(P=0.0003)and protein(P=0.0076)expression of ERAP1 was significantly increased compared to the transfected null group,indicating that the wild-type plasmid acted as an overexpressor of the target gene.After transfection with variant plasmids ERAP1-c.1583T>C and ERAP1-c.2188G>C,the m RNA expression of the target gene was significantly decreased(c.2188G>C: P=0.0003;c.1583T>C: P=0.0004)and the protein expression was significantly decreased(c.2188G>C: P= 0.0033;c.1583T>C: P=0.006),indicating that these two missense variants were able to down-regulate the expression of the target genes.4.After overexpression of the target gene in cells: the proliferation ability of cells was enhanced(P<0.0001);the migration rate of cells increased after 48 h of scratching(P=0.0015)and the number of cells crossing transwell chambers increased at24h(P=0.001),indicating enhanced migration ability of cells;transwell invasion assay showed enhanced invasion ability of cells(P= 0.0001).After transfection of both missense variant plasmids into cells,compared with the overexpression group: the proliferation ability of cells was inhibited(c.2188G>C: P<0.0001;c.1583T>C: P<0.0001);the number of transwell cells crossing was significantly decreased(c.2188G>C: P=0.0013;c.1583T>C: P= 0.0318),and the cell migration rate all showed a decreasing trend after48 h of scratching,with a statistically different decreasing trend for transfected ERAP1-c.2188G>C plasmid(P=0.0043)and a non-significant decreasing trend for transfected ERAP1-c.1583T>C plasmid(P=0.0985);the invasive ability of cells decreased(c.2188G>C: P < 0.001;c.1583T>C: P=0.0227).Conclusion: The high expression of ERAP1 enhances cell proliferation,migration and invasion,while two missense variants,ERAP1-c.1583T>C and ERAP1-c.2188G>C,reduce the expression of the target gene and thus inhibit the cell function.We therefore hypothesized that ERAP1 as a genetic susceptibility factor for pre-eclampsia may be involved in the pathogenesis of pre-eclampsia through its locus polymorphisms that affect the migratory invasive ability of trophoblast cells. |
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