WNT3 hypomethylation counteracts low activity of the Wnt signaling pathway in the placenta of preeclampsia

Abstract

Preeclampsia is a hypertensive disorder of pregnancy. Many studies have shown that epigenetic mechanisms may play a role in preeclampsia. Moreover, our previous study indicated that the differentially methylated genes in preeclampsia were enriched in the Wnt/β-catenin signaling pathway. This study aimed to identify differentially methylated Wnt/β-catenin signaling pathway genes in the preeclamptic placenta and to study the roles of these genes in trophoblast cells in vitro. Using an Illumina Infinium HumanMethylation 850 K BeadChip, we found that the Wnt signaling pathway was globally hypermethylated in the preeclamptic group compared with the term birth group, but hypomethylated in the preeclamptic group compared with the preterm birth group. Among all Wnt/β-catenin signaling pathway factors, WNT3 was the most significantly differentially expressed gene and was hypomethylated in the preeclamptic group compared to the nonhypertensive groups, namely, the preterm birth group and term birth group. This result was confirmed by pyrosequencing. Through quantitative real-time PCR and western blot analysis, the WNT3 gene was found to be highly expressed in preeclamptic placental tissues, in contrast to other WNT factors, which were previously reported to be expressed at low levels in placental tissues. Additionally, in the HTR8/SVneo cell line, knockdown of WNT3 suppressed the Wnt/β-catenin signaling pathway, consistent with the findings for other WNT factors. These results prompted us to speculate that the WNT3 gene counteracts the low activation state of the Wnt signaling pathway in the preeclamptic placenta through methylation modification.

Keywords: DNA methylation; Placentas; Preeclampsia; Preterm birth; WNT3 gene.

Fig. 1

The canonical Wnt/β-catenin pathway. A Without Wnt ligand, β-catenin in the cytoplasm is phosphorylated and then degraded by ubiquitination. B With Wnt ligand, phosphorylated GSK3β and β-catenin increase in the cytoplasm, and the latter enters the nucleus to regulate gene expression. LRP lipoprotein receptor-related protein, Dvl disheveled, APC adenomatous polyposis coli, GSK3β glycogen synthase kinase 3β, CK1 casein kinase 1, TCT/LEF T-cell factor/lymphocyte enhancer binding factor

Fig. 2

The global methylation level of Wnt signaling pathway among three groups detected by Illumina 850 K Beadchip. A Comparison between TB and PE; B Comparison between PE and PB; C Comparison between TB and PB. Blue color represents hypomethylated sites, while red color represents hypermethylated sites. TB term birth, PB preterm birth, PE preeclampsia. β value represents the methylation levels of detected sites

Fig. 3

The methylation levels of WNT3 gene by pyrosequencing. A The average levels of the four CpGs in PE (12.4% ± 2.2%) compared with TB (14.5% ± 2.2%) and PB (14.3% ± 2.5%) groups. B–E The methylation levels of the four CpGs. TB term birth, PB preterm birth, PE preeclampsia. Unpaired t test. *P < 0.05, **P < 0.01

Fig. 4

The expression levels of WNT3 gene in placentas of the three group and its correlation with methylation levels. A The mRNA expression levels of WNT3 in placentas in the three groups. B The correlation between mRNA expression and methylation levels. r = − 0.525, R² = 0.2071, P < 0.05. C, D The relative protein expression of WNT3 in placentas of the three groups by western blotting. TB term birth, PB preterm birth, PE preeclampsia. Unpaired t test, Welch's t test, Mann–Whitney test, and Pearson correlation analysis. *P < 0.05, **P < 0.01. ***P < 0.001, ****P < 0.0001

Fig. 5

Immunostaining of WNT3 protein in placental tissue sections of the three groups WNT3 protein was located in the villous trophoblasts (VT) and extravillous trophoblasts (EVT). TB term birth, PB preterm birth, PE preeclampsia. Original magnification 400 × for A, C, E, 200 × for B, D and F

Fig. 6

The mRNA expression levels of WNT3 after demethylation by 5-Aza-dC in JAR and HTR8/Svneo cell lines. A The expression of WNT3 in JAR cell line. B The expression of WNT3 in HTR8/Svneo cell line. 5-Aza-dC: 5-Aza-2'-deoxycytidine. Unpaired t test and Mann–Whitney test. *P < 0.05; **P < 0.01

Fig. 7

The shRNA sequence of WNT3 gene

Fig. 8

Loss of WNT3 suppressed the Wnt/β-catenin signaling pathway. A Expression of Wnt/β-catenin signaling pathway proteins in HTR8/SVneo cell line after transfection with shRNA. 1–3, Control; 4–6, sh-WNT3 (cells were transfected with shRNA-WNT3); 7–9, sh-NC. B The relative protein expression of WNT3, β-catenin, phosphorylated β-catenin, GSK3β, phosphorylated GSK3β, and ratio of phosphorylated and non-phosphorylated protein in HTR8/SVneo cell line. One-Way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 9

WNT3 promoted trophoblast cell proliferation. A Cell Counting Kit-8 assay was used to detect the proliferation of HTR8/SVneo cells. B Cell proliferation increased after transfection with WNT3-plasmid in HTR8/SVneo cells. One-way ANOVA. *P < 0.05, **P < 0.01. Image acquisition tools: IBM SPSS Statistics 25 and GraphPad Prism 8.4.2. Image processing software packages: Adobe photoshop cs6 13.0.1