Amnion-Derived Mesenchymal Stem Cell Exosomes-Mediated Autophagy Promotes the Survival of Trophoblasts Under Hypoxia Through mTOR Pathway by the Downregulation of EZH2

Abstract

Human amnion-derived mesenchymal stem cells (AD-MSCs) have been reported as a promising effective treatment to repair tissue. Trophoblast dysfunction during pregnancy is significantly involved in the pathogenesis of preeclampsia (PE). To understand how AD-MSCs regulated trophoblast function, we treated trophoblasts with AD-MSC-derived exosomes under hypoxic conditions. The treatment markedly enhanced the trophoblast proliferation and autophagy. Furthermore, significant decrease of EZH2 levels and inactivation of mTOR signaling were observed in AD-MSC exosomes-treated trophoblasts. Consistent with these findings, overexpression of EZH2 activated the mTOR signaling in trophoblasts, and reduced the autophagy and survival of trophoblasts, even in the presence of AD-MSC-derived exosomes. In addition, EZH2 inhibition exhibited the same trophoblast autophagy-promoting effect as induced by AD-MSC-derived exosomes, also accompanied by the inactivation of mTOR signaling. Importantly, when EZH2 was overexpressed in trophoblasts treated with PQR620, a specific mTOR signaling inhibitor, the autophagy and proliferation in trophoblasts were decreased. Studies on human placental explants also confirmed our findings by showing that the expression levels of EZH2 and mTOR were decreased while the autophagy-associated protein level was increased by AD-MSC-derived exosome treatment. In summary, our results suggest that EZH2-dependent mTOR signaling inactivation mediated by AD-MSC-derived exosomes is a prerequisite for autophagy augmentation in hypoxic trophoblasts.

Keywords: EZH2; autophagy; chorionic villous-derived mesenchymal stem cells; mTOR signaling; trophoblasts.

FIGURE 1

AD-MSC-derived exosomes promote the autophagy and proliferation of trophoblasts in hypoxia condition. (A) Representative CCK-8 assay results for JEG-3 and HTR-8 cells are shown. Trophoblast cells were treated with AD-MSC exosomes under hypoxic conditions. (B) Representative EdU assay results for trophoblasts are shown. The histogram of EdU positive trophoblasts that treated with AD-MSC exosomes under hypoxic conditions was shown. (C) Whole cell lysates from trophoblast cells were subjected to western blotting to analyze and quantificate LC3, BECN1 and p62 levels. β-actin was included as a loading control. (D) Immunofluorescence analysis showed autophagosomes in the cytoplasm of trophoblast cells treated with AD-MSC exosomes under hypoxic conditions (scale bar, 25 μm). (E) Significant decreases in EZH2, mTOR and S6K1 mRNA levels were found in JEG-3 and HTR-8 cells treated with AD-MSC exosomes by qRT-PCR. (F) Whole cell lysates from trophoblast cells were subjected to western blotting to analyze EZH2, mTOR, S6K1, p-mTOR and p-S6K1 levels and quantificate EZH2 expression. β-actin was included as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 2

The inhibition of EZH2 induced the increase of trophoblast autophagy and inhibition of mTOR pathway under hypoxic conditions. (A–C) The protein expression level of LC3, BECN1 and P62 levels were examined in JEG-3 and HTR-8 cells treated with GSK126, AD-MSC exosomes or Baf A1 for 24 h by western blot. The protein expression levels were quantified by densitometry. (D) Cell proliferation was evaluated by a CCK-8 assay. The trophoblast cell lines were treated with GSK126 or Baf A1 and with or without AD-MSC exosomes under hypoxic conditions. (E) The EdU positive percent of trophoblast cell lines were tested under hypoxic conditions by flow cytometry. **P < 0.01, *P < 0.05, ***P < 0.001.

FIGURE 3

The knockdown of EZH2 induced the increase of trophoblast autophagy and mTOR pathway inhibition under hypoxic conditions. (A) Significant decreases in EZH2, mTOR and S6K1 mRNA levels were tested in JEG-3 and HTR-8 cells transfected with EZH2 siRNA by qRT-PCR. (B,C) The trophoblast cell lines were transfected with a EZH2 siRNA under hypoxic conditions, and BECN1, P62 and LC3 expression levels were tested by western blotting analysis. Empty vector (scramble) cells served as controls. (D,E) Cell proliferation was evaluated by a CCK-8 assay and EdU flow cytometry. The two trophoblast cell lines were transfected with EZH2 siRNA with or without AD-MSC exosomes treatment under hypoxic conditions. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 4

EZH2 overexpression decreased trophoblast autophagy and proliferation through mTOR signaling pathway. (A) Significant increases in EZH2, mTOR and S6K1 mRNA were found in JEG-3 and HTR-8 cells transfected with EZH2 overexpression plasmid (EZH2) compared to empty vector (Vec)-transfected cells under hypoxic conditions by qRT-PCR. (B,C) The two trophoblast cell lines were transfected with EZH2 overexpression plasmid under hypoxic conditions, and BECN1, P62 and LC3 protein levels were tested by western blotting analysis. (D,E) Cell proliferation was evaluated by a CCK-8 assay and EdU flow cytometry. The trophoblast cell lines were transfected with a EZH2 overexpression plasmid or empty vector under hypoxic conditions. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 5

EZH2 regulate trophoblast autophagy and proliferation through mTOR signaling pathway. (A) The levels of EZH2, p-mTOR, p-S6K1, and autophagy associated proteins were examined in JEG-3 and HTR-8 cells transfected with EZH2 overexpression plasmid and treated with m-TOR inhibitor PQR620 under hypoxic conditions by western blot. (B,C) Cell proliferation was evaluated by a CCK-8 assay and EdU flow cytometry. The trophoblast cell lines were treated with EZH2 overexpression plasmid and m-TOR inhibitor PQR620 under hypoxic conditions. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 6

AD-MSC-mediated EZH2 activation increased autophagy in placental explants under hypoxic conditions. (A) Placental explants were treated with AD-MSC exosomes transfected with EZH2 overexpression plasmids or Vec under hypoxic conditions, and EZH2, LC3 expression levels were tested by western blotting analysis. (B) Placental explants were treated with AD-MSC exosomes under hypoxic conditions, and EZH2 were tested by immunohistochemistry. Untreated placental explants served as controls (scale bar, 50 μm). *P < 0.05, **P < 0.01.