ABSTRACT
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 1 240 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: χ2=29.25, Allele: χ2=4.68) and rs469783 (Genotype: χ2=7.01, Allele: χ2=6.45) as well as the genotype distribution of rs27044 ( χ2=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 ( χ2=20.82 and 19.97, both P<0.05), but a lower frequency in CC dominant gene pattern for rs469783 ( χ2=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.