Long noncoding RNA maternally expressed gene 3 improves trophoblast dysfunction and inflammation in preeclampsia through the Wnt/ β-Catenin/nod-like receptor pyrin domain-containing 3 axis

Abstract

Inadequate trophoblastic infiltration and resulting placental hypoxia and inflammation comprise the core pathological basis of preeclampsia (PE). Maternally expressed gene 3 (MEG3) is known to be involved in the pathogenesis of preeclampsia by inhibiting the migration and invasion of trophoblasts and promoting their apoptosis. Nevertheless, the specific underlying downstream molecular mechanism of MEG3 is less well characterized. In this study, we detected lower expression levels of MEG3 and β-Catenin and higher expression of nod-like receptor pyrin domain-containing 3 (NLRP3) in placental tissues of pregnant women with severe preeclampsia (sPE) than in normal pregnancies. Elevated serum levels of IL-1β and TNF-α were also observed in the sPE group. Then, we established a hypoxia/reoxygenation (H/R) model to mimic preeclampsia. Similar results with sPE group were found in the H/R group compared with the control group. In addition, suppressive trophoblast proliferation, migration and invasion and increases in the apoptotic rate and inflammation were also detected in the H/R group. Notably, overexpressing MEG3 markedly improved trophoblast dysfunction and inflammation caused by H/R. However, the effects of MEG3 on trophoblasts, whether upregulated or downregulated, can be reversed by DKK-1 (Wnt/β-Catenin inhibitor) and MCC950 (NLRP3 inhibitor). The current study revealed that MEG3 regulates trophoblast function and inflammation through the Wnt/β-Catenin/NLRP3 axis and provided new insights into the pathogenesis of preeclampsia.

Keywords: NLRP3; Wnt/β-Catenin; inflammation; lncRNA MEG3; placenta; preeclampsia; trophoblast.

FIGURE 1

Expression of MEG3, β-Catenin and NLRP3 in the placentas of normal pregnancies and sPE patients. The relative mRNA levels of MEG3 (A), β-Catenin (B) and NLRP3 (C) were assessed by qRT‒PCR. (D,E) The relative protein levels of β-Catenin and NLRP3 were detected by Western blot. GAPDH was used as an endogenous control. The expression levels of NLRP3 (F) and β-Catenin (G) in placental tissues were evaluated by immunofluorescence (IF). (H) Semi-quantitative analysis of mean fluorescence intensity. The serum levels of IL-1β (I) and TNF-α (J) were detected by ELISA. n = 20/group. **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 2

H/R treatment affected the expression of MEG3, β-Catenin and NLRP3 in trophoblast cells. H/R-induced changes in MEG3 (A), β-Catenin (B) and NLRP3 (C) mRNA levels were assessed by qRT‒PCR (n = 7/group). (D,E) H/R-induced changes in β-Catenin and NLRP3 protein levels were detected by Western blot (n = 3/group). (F,G) The expression levels of c-Myc, Vimentin, Bcl-2, and Bax induced by H/R were detected by Western blotting (n = 3/group). GAPDH was used as an endogenous control. *p < 0.05; **p < 0.01.

FIGURE 3

H/R-induced trophoblast dysfunction and inflammation were used to mimic preeclampsia. (A) Cell proliferation was determined by CCK-8 assay. (B,C) Cell invasion ability was determined by transwell assay. (D,E) Cell migration ability was determined by scratch assay. (F,G) The apoptosis rate was measured by flow cytometry. ELISA was used to detect IL-1β (H) and TNF-α (I) in the cell supernatant. n = 3/group. **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 4

The overexpression of MEG3 altered the expression levels of β-Catenin and NLRP3 in trophoblast cells. The mRNA expression levels of MEG3 (A), β-Catenin (B) and NLRP3 (C) after overexpression of MEG3 were assessed by qRT‒PCR (n = 4/group). (D,E) The protein expression levels of β-Catenin and NLRP3 after overexpression of MEG3 were detected by Western blot (n = 3/group). (F,G) The expression levels of c-Myc, Vimentin, Bcl-2, and Bax were assessed by Western blotting (n = 3/group). GAPDH was used as an endogenous control. *p < 0.05; **p < 0.01; ****p < 0.0001.

FIGURE 5

The overexpression of MEG3 overcame H/R-induced trophoblast dysfunction and inflammatory cytokine secretion. (A) Cell proliferation was determined by CCK-8 assay. (B,C) Cell invasion ability was determined by transwell assay. (D,E) Cell migration ability was determined by scratch assay. (F,G) The apoptosis rate was measured by flow cytometry. ELISA was used to measure IL-1β (H) and TNF-α (I) levels in the cell supernatant. n = 3/group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 6

Blocking Wnt/β-Catenin signaling reversed the effect of MEG3 on the expression levels of β-Catenin and NLRP3. qRT‒PCR analysis of β-Catenin (A) and NLRP3 (B) in HTR8/SVneo cells overexpressing MEG3 treated with DKK-1 (n = 4/group). (C,D) Western blot analysis of β-Catenin and NLRP3 in HTR8/SVneo cells overexpressing MEG3 treated with DKK-1 (n = 3/group). (E,F) Western blot analysis of the expression levels of c-Myc, Vimentin, Bcl-2, and Bax (n = 3/group). GAPDH was used as an endogenous control. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 7

Blocking Wnt/β-Catenin signaling disrupted the effect of MEG3 on trophoblast function. (A) Cell proliferation was determined by CCK-8 assay. (B,C) Cell invasion ability was determined by transwell assay. (D,E) Cell migration ability was determined by scratch assay. (F,G) The apoptosis rate was measured by flow cytometry. ELISA was used to measure IL-1β (H) and TNF-α (I) levels in the cell supernatant. n = 3/group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 8

Suppressing NLRP3 expression reversed the effect of MEG3 knockdown on the expression level of NLRP3. qRT‒PCR [(A); n = 4/group] and Western blotting [(B,C); n = 3/group] were performed to evaluate NLRP3 mRNA and protein levels in MEG3 knockout HTR8/SVneo cells treated with MCC950. (D,E) Western blotting was performed to evaluate the expression levels of c-Myc, Vimentin, Bcl-2, and Bax (n = 3/group). GAPDH was used as an endogenous control. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 9

Suppressing NLRP3 expression alleviated the effect of MEG3 knockdown on trophoblast function. (A) Cell proliferation was determined by CCK-8 assay. (B,C) Cell invasion ability was determined by transwell assay. (D,E) Cell migration ability was determined by scratch assay. (F,G) The apoptosis rate was measured by flow cytometry. ELISA was used to measure IL-1β (H) and TNF-α (I) levels in the cell supernatant. n = 3/group. *p < 0.05; **p < 0.01; ****p < 0.0001.