| Introduction—Increasing evidence suggests that an exaggerated maternal systemic inflammatory response may play a central role in the pathogenesis of preeclampsia(PE).Considering the growing evidence on microRNAs(miRNA)and tissue-specific regμLators of gene expression,we investigated the potential association of miR-210 and Foxp3 in preeclamptic patients.The aim of this study was to investigate whether single nucleotide polymorphisms(SNPs)in the genes that encode forkhead boxP3(Foxp3)(rs3761549 OT,rs2280883T>C,rs2232365 A>G and rs3761548 OA)and transforming growth factor(TGF)-β1(rs11466359 OT,rs11466345 A>G and rs1800469 T>C)are associated with preeclampsia(PE)risk in Chinese women.Methods—Serum levels of cytokines(IL-6,IL-10,IL-17,and TGF-β1)were detected with enzyme-linked immunosorbent assay(ELISA).29 patients with late□onset preeclampsia(≥36 weeks of gestation),27 pregnant women with normal uncomplicated pregnancies(≥36 weeks of gestation)and 20 healthy non□pregnant women.Reverse-transcription polymerase chain reaction(RT-PCR)was performed to detect mRNA expression for maternal placenta RORc,Foxp3,and miR-210.Foxp3 protein expression was evaluated by Western blot.The study included 29 patients with PE with placenta as the experimental group and 27 women with normal pregnancy placenta as a control group.Bioinformatics methods such as mirbase/targetscans/pictartar were used to predict the target genes of mir-210 and luciferase reporter gene analysis was used to verify the potential target genes.SNPs were identified by polymerase chain reaction(PCR)and ligase detection reaction(LDR).Allelic variant and genotype frequencies for Foxp3 and TGF-β1 were compared between PE women(N = 203)and healthy pregnant(HP)controls(N =243).Results—① Plasma production of IL□6,IL□17 and TGF□β1 was significantly higher in preeclamptic patients compared with these levels in normal pregnant women(P=0.0021,P=0.015 and P=0.019,respectively).Accordingly,levels of Treg cytokines such as IL□10 were significantly lower in preeclamptic women than in normal pregnant women(P=0.0012).② The percentage of CD4+CD25+CD127-cells among the CD4+ T cells was analyzed in peripheral blood of patients,and the percentage of CD4+CD25+CD127-cells among the CD4+ T cells was significantly lower in the peripheral blood of patients with PE compared with normal pregnant women(6.34±0.28 vs.9.10±0.28%;P<0.001).The percentage of CD4+CD25+CD127-cells among the CD4+ T cells was significantly lower in the peripheral blood of PE compared with healthy nonDpregnant women(6.34±0.28 vs.8.75±0.54%;P<0.001).③ The results demonstrated that mRNA expression of Foxp3 was significantly lower in preeclamptic women compared with normal pregnant women(0.713±0.088 vs.1.105±0.136;P=0.019),In addition,mRNA expression levels of RORc were significantly higher in women with PE compared with normal pregnant women(0.599±0.069 vs.0.380±0.046;P=0.0127).④ In the present study,significantly higher expression of miR□210 was observed in patients with PE compared with normal pregnant women(1.744±0.153 vs.0.578±0.744;P<0.01).Consistent with mRNA expression results,lower protein expression levels of Foxp3 was observed in patients with PE compared with normal pregnant subjects.⑤According to the prediction of bioinformatics and the analysis of luciferase reporter gene,we confirmed that Foxp3 is the target gene of mir-210,the key transcription factor of regulatory t cells.⑥ In this study,a reporter gene expression vector containing the binding site of Foxp3 gene 3’-UTR miR-210 was constructed.The results of luciferase reporter system assay showed that the luciferase reporter gene expression was significantly decreased in psiCHECK-2-REPORT-Foxp3WT compared with that in the co-transfection group with miR-210 mimic and the negative control group,while the luciferase reporter gene expression was not significantly decreased in the psiCHECK-2-REPORT-Foxp3Mut compared with that in the transfection group with mir-210 mimic and the negative control group.It was further proved that Foxp3 was the target gene of miR-210.⑦ The effect of overexpression of miR-210 on the expression of the regulatory T cell transcription factor Foxp3 was compared with that of the control group.Westernblot assay of Foxp3 protein levels in CD4+T cells transfected with miR-210 mimic showed significant reduction,and the semi-quantitative analysis of gray scale values showed significant differences.The difference was statistically significant(P<0.05).⑧ The rs1800469 TT genotype was found more frequently in PE patients than in HP controls[CC vs.CT+TT:odds ratio(OR)=1.71,95%CI,1.04-2.81;p =0.033],indicating that the T allele of rs1800469 confers a risk for PE[OR = 1.46;95%CI,1.12-1.92;p= 0.006].Conclusion—Our data suggest that PE manifests as a decreased number of Tregs,which regulate maternal tolerance of the fetus.In placenta from women with PE,compared with normal pregnant women,mRNA expression of Foxp3 was significantly decreased,and expression of miR-210 was significantly increased.According to the prediction of bioinformatics and the analysis of luciferase reporter gene,we confirmed that Foxp3 is the target gene of miR-210,the key transcription factor of regulatory t cells.The present findings support that allelic variants of TGF-β1 rs1800469 T influence PE risk in Chinese women.Pregnant Han Chinese women carrying the rs 1800469 TT genotype were at increased risk of PE. |
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