内质网氨基肽酶1与子痫前期遗传易感性研究

Endoplasmic reticulum aminopeptidase 1 and genetic susceptibility to pre-eclampsia

目的探究内质网氨基肽酶1(endoplasmic reticulum aminopeptidase 1,ERAP1)是否为子痫前期发病的遗传易感基因,以及ERAP1参与子痫前期发病的分子机制。方法回顾性选择2018年9月至2021年4月在青岛大学附属医院、枣庄市妇幼保健院等6家山东省地级市三甲医院的990例子痫前期患者(病例组)和1240例健康孕妇(对照组)。收集所有孕妇的外周血样本,提取DNA,通过Taqman探针聚合酶链反应技术进行rs30187、rs27044及rs469783位点的基因分型;并在构建ERAP1野生型质粒的基础上使用点突变法构建2种错义变异质粒:rs30187(c.1583A>G)、rs27044(c.2188C>G),将不同的转染分子转染至Htr8细胞中,检测ERAP1的表达水平,并通过Transwell迁移实验、Transwell侵袭实验、细胞划痕实验和细胞增殖实验检测不同转染对细胞功能的影响,根据转染分子的不同,分为以下6组:敲低对照组、敲低组、过表达对照组、过表达组、变异1组及变异2组。采用两独立样本t检验、秩和检验和χ^(2)检验进行统计学分析。结果(1)rs30187位点的遗传学分布(基因型:χ^(2)=29.25;等位基因:χ^(2)=4.68)、rs27044位点的基因型分布(χ^(2)=28.95)、rs469783位点的遗传学分布(基因型:χ^(2)=7.01,等位基因:χ^(2)=6.45)在病例组和对照组间差异有统计学意义(P值均<0.05)。病例组中rs30187和rs27044位点均是隐性基因CC的频率更高(χ^(2)值分别为20.82和19.97,P值均<0.05),而rs469783位点的显性基因中CC基因型的频率更低(χ^(2)=5.82,P=0.016)。(2)细胞转染后,与敲低对照组相比,敲低组ERAP1的表达水平明显下降(mRNA:0.5±0.1与1.0±0.0,t=7.49;蛋白:0.4±0.1与0.7±0.1,t=2.81;P值均<0.05);划痕48 h后,敲低组细胞迁移率明显下降[(16.5%±1.8%)与(23.8%±2.4%),t=3.33,P=0.031];细胞培养24 h后,敲低组Transwell细胞数量明显下降(423.7±21.3与499.0±24.6,t=3.29,P=0.031)。转染变异1组和变异2组后,与过表达组相比,ERAP1的mRNA和蛋白表达也均明显下降,Transwell小室细胞穿过的数量明显下降,划痕48 h后细胞迁移率均表现出下降趋势[变异1组:P=0.004,变异2组:(21.1±4.6)%与(28.3±1.1)%,t=2.10,P=0.099]。转染过表达组至细胞后,与过表达对照组相比,ERAP1的mRNA和蛋白表达明显上升,细胞的增殖能力增强,细胞划痕48 h后迁移率升高,24 h穿过Transwell小室的细胞数量增多,细胞侵袭能力增强(P值均<0.05)。结论ERAP1基因rs30187、rs27044和rs46978与子痫前期易感性相关,rs30187和rs27044位点上子痫前期患者CC基因型携带者更多,而rs469783位点上健康孕妇CC基因型的携带者更多。ERAP1可能通过影响滋养层细胞的迁移侵袭能力参与子痫前期的发病。

Objective To investigate whether endoplasmic reticulum aminopeptidase 1(ERAP1)is a susceptible gene for pre-eclampsia(PE)and the possible mechanism in the pathogenesis.Methods This retrospective study included 990 PE patients(case group)and 1240 healthy pregnant women(control group)in six prefecture-level tertiary hospitals in Shandong Province,including the Affiliated Hospital of Qingdao University and Zaozhuang Maternal and Child Health Hospital,from September 2018 to April 2021.Peripheral blood were collected for DNA extraction.Single-nucleotide polymorphisms in the ERAP1 gene(rs30187,rs27044,and rs469783 loci)were analyzed by Taqman probe polymerase chain reaction(PCR).Two missense mutant plasmids,rs30187(c.1583A>G)and rs27044(c.2188C>G),were constructed by point mutation induction based on wild-type plasmids.Six groups(knock-down control,knock-down,over-expression control,over-expression,variant 1 and 2 groups)were set up in this study.After transfecting Htr8 cells with different transfection molecules,the expression of ERAP1 at mRNA and protein levels were detected.Besides,the effects of different transfections on cell function were detected using Transwell migration assay,Transwell invasion assay,cell scratch assay,and CCK-8 assay.Statistical analysis was performed using two independent samples t-test,rank sum test,and Chi-square test.Results(1)There were significant differences in the genetic distribution of rs30187(Genotype:χ^(2)=29.25,Allele:χ^(2)=4.68)and rs469783(Genotype:χ^(2)=7.01,Allele:χ^(2)=6.45)as well as the genotype distribution of rs27044(χ^(2)=28.95)between the case group and the control group(all P<0.05).Statistical analysis of the genetic model revealed that rs30187 and rs27044,both recessive models,were statistically different between the two groups with a higher frequency of CC genotypes in the case group(χ^(2)=20.82 and 19.97,both P<0.05),but a lower frequency in CC dominant gene pattern for rs469783(χ^(2)=5.82,P=0.016).(2)Compared with the knock-down control group,the knock-down group showed significantly inhibited expression of ERAP1(mRNA:0.5±0.1 vs 1.0±0.0,t=7.49;protein:0.4±0.1 vs 0.7±0.1,t=2.81;both P<0.05),reduced cell migration rate after 48 h of scratching[(16.5%±1.8%)vs(23.8%±2.4%),t=3.33,P=0.031]and decreased number of cells crossing Transwell chambers after 24 h of culture(423.7±21.3 vs 499.0±24.6,t=3.29,P=0.031).Compared with the over-expression group,variant 1 group and variant 2 group showed significantly inhibited expression of ERAP1 at mRNA(both P<0.001)and protein(P=0.003 and 0.006)levels after transfection,decreased number of cells crossing Transwell chambers(P=0.001 and 0.032)and down-regulated cell migration rate after 48 h of scratching[variant 1:P=0.004;variant 2:(21.1±4.6)%vs(28.3±1.1)%,t=2.10,P=0.099].ERAP1 expression at both mRNA(P<0.001)and protein(P=0.008)levels,as well as cell proliferation(P<0.001)and invasion ability(P<0.001),were all enhanced in the over-expression group than those in the over-expression control group.Moreover,the migration rate of cells after 48 h of scratching(P=0.002)and the number of cells crossing Transwell chambers after 24 h of culture(P=0.001)were also increased.Conclusions The rs30187,rs27044,and rs46978 on ERAP1 gene were all associated with PE susceptibility,with more carriers of the CC genotype in PE patients at rs30187 and rs27044 loci and more carriers of the CC genotype in healthy gravida at rs469783 locus.ERAP1 may be involved in the pathogenesis of PE by affecting the migratory and invasive ability of trophoblast cells.