The role of Zeb1 in the pathogenesis of morbidly adherent placenta

Abstract

Zinc finger E‑box‑binding homeobox 1 (Zeb1) is a promoter of epithelial‑mesenchymal transformation, which may serve an important role in morbidly adherent placenta (MAP). In the present study, the protein expression levels of Zeb1 were examined in the placenta tissues of 60 patients, including 20 patients with placenta accreta (PA) and 20 patients with placenta previa without PA (UPA) and 20 patients in late pregnancy that delivered by cesarean section (normal). The expression levels of Zeb1, N‑cadherin, vascular endothelial growth factor (VEGF), Tumor necrosis factor‑related apoptosis‑inducing ligand‑receptor 2 (TRAIL‑R2), and tumor necrosis factor‑related apoptosis‑inducing ligand‑receptor 3 (TRAIL‑R3) were higher in PA tissues compared with in normal control tissues. The expression levels of E‑cadherin and TRAIL‑R2 were decreased in PA tissues compared with in normal control tissues. These findings indicated that Zeb1 may serve an important role in placental attachment, thus promoting the development of dangerous PA. Overexpression of Zeb1 may upregulate the expression levels of N‑cadherin, VEGF, TRAIL‑R3, cyclin D1 and Bcl‑2, and downregulate the expression levels of E‑cadherin and TRAIL‑R2. In addition, Zeb1 regulated the viability, apoptosis and migration of HTR‑8/SV neo cells and human umbilical vein endothelial cells by regulating the Akt pathway. In conclusion, these findings indicated that Zeb1 may promote placental implantation by activating the Akt signaling pathway, thus providing a theoretical basis for investigating the causes of MAP.

Figure 1.

Zeb1 expression in placenta tissues. Immunohistochemical staining for the expression of (A) Zeb1 and (B) VEGF in placenta tissues (magnification, ×100 and ×400). **P<0.05 vs. PA tissues. E-cad, E-cadherin; N-cad, N-cadherin; PA, placenta accrete; TRAIL-R, TNF receptor superfamily member; UPA, placenta previa without PA; VEGF, vascular endothelial growth factor; Zeb1, zinc finger E-box-binding homeobox 1. Zeb1 expression in placenta tissues. Immunohistochemical staining for the expression of (C) E-cad in placenta tissues (magnification, ×100 and ×400). **P<0.05 vs. PA tissues. (D and E) Expression of Zeb1, E-cad, N-cad, VEGF, TRAIL-R2 and TRAIL-R3 in placenta tissues was detected by western blot analysis and reverse transcription-quantitative PCR **P<0.05 vs. PA tissues; ^##P<0.05 vs. UPA tissues. E-cad, E-cadherin; N-cad, N-cadherin; PA, placenta accrete; TRAIL-R, TNF receptor superfamily member; UPA, placenta previa without PA; VEGF, vascular endothelial growth factor; Zeb1, zinc finger E-box-binding homeobox 1.

Figure 1.

Zeb1 expression in placenta tissues. Immunohistochemical staining for the expression of (A) Zeb1 and (B) VEGF in placenta tissues (magnification, ×100 and ×400). **P<0.05 vs. PA tissues. E-cad, E-cadherin; N-cad, N-cadherin; PA, placenta accrete; TRAIL-R, TNF receptor superfamily member; UPA, placenta previa without PA; VEGF, vascular endothelial growth factor; Zeb1, zinc finger E-box-binding homeobox 1. Zeb1 expression in placenta tissues. Immunohistochemical staining for the expression of (C) E-cad in placenta tissues (magnification, ×100 and ×400). **P<0.05 vs. PA tissues. (D and E) Expression of Zeb1, E-cad, N-cad, VEGF, TRAIL-R2 and TRAIL-R3 in placenta tissues was detected by western blot analysis and reverse transcription-quantitative PCR **P<0.05 vs. PA tissues; ^##P<0.05 vs. UPA tissues. E-cad, E-cadherin; N-cad, N-cadherin; PA, placenta accrete; TRAIL-R, TNF receptor superfamily member; UPA, placenta previa without PA; VEGF, vascular endothelial growth factor; Zeb1, zinc finger E-box-binding homeobox 1.

Figure 2.

si-Zeb1 inhibits the growth and migration of HTR-8/sv neo cells. (A) Viability of HTR-8/sv neo cells transfected with scrambled siRNA or si-Zeb1 was assessed at different time points. (B) Flow cytometric analysis of cell cycle progression of HTR-8/sv neo cells transfected with scrambled siRNA or si-Zeb1 for 24 h. (C) Flow cytometric analysis of apoptosis of HTR-8/sv neo cells transfected with scrambled siRNA or si-Zeb1 for 24 h. The Q3 region contains early apoptotic cells; these data were used for statistical analysis. (D) Migration of HTR-8/sv neo cells transfected with scrambled siRNA or si-Zeb1 for 24 h (magnification, ×400). (E and F) Expression levels of Zeb1, E-cad, N-cad, VEGF, TRAIL-R2, TRAIL-R3, cyclin D1 and Bcl-2, as detected by western blot analysis and reverse transcription-quantitative PCR, in HTR-8/sv neo cells transfected with scrambled siRNA or si-Zeb1 for 24 h. All experiments were repeated in triplicate **P<0.05 compared with scrambled control. E-cad, E-cadherin; N-cad, N-cadherin; OD, optical density; si/siRNA, small interfering RNA; TRAIL-R, TNF receptor superfamily member; VEGF, vascular endothelial growth factor; Zeb1, zinc finger E-box-binding homeobox 1.

Figure 3.

HUVEC viability and migration are inhibited by si-Zeb1. (A) Results of MTT viability assays in HUVECs transfected with scrambled siRNA or si-Zeb1 at different time points. (B) Flow cytometric analysis of cell cycle progression of HUVECs transfected with scrambled siRNA or si-Zeb1 for 24 h. (C) Flow cytometric analysis of apoptosis of HUVECs transfected with scrambled siRNA or si-Zeb1 for 24 h. The Q3 region contains early apoptotic cells; these data were used for statistical analysis. (D) Migration of HUVECs transfected with scrambled siRNA or si-Zeb1 for 24 h (magnification, ×400). (E and F) Expression of Zeb1, E-cad, N-cad, VEGF, TRAIL-R2, TRAIL-R3, cyclin D1 and Bcl-2, as detected by western blot analysis and reverse transcription-quantitative PCR, in HUVECs transfected with scrambled siRNA or si-Zeb1 for 24 h. All experiments were repeated in triplicate **P<0.05 compared with scrambled control. E-cad, E-cadherin; HUVECs, human umbilical vein endothelial cells; N-cad, N-cadherin; OD, optical density; si/siRNA, small interfering RNA; TRAIL-R, TNF receptor superfamily member; VEGF, vascular endothelial growth factor; Zeb1, zinc finger E-box-binding homeobox 1.

Figure 4.

Zeb1 promotes cell growth and migration through the Akt pathway. (A and B) Western blotting and reverse transcription-quantitative PCR analysis of Zeb1, E-cad, N-cad, VEGF, TRAIL-R2, TRAIL-R3, Akt, Akt^p-Ser473, Akt^p-Tyr308, cyclin D1 and Bcl-2 expression in HTR-8/sv neo cells in the si-Zeb1, si-Zeb1 + IGF1 or scrambled groups. **P<0.05 compared with scrambled group; ^##P<0.05 compared with si-Zeb1 group. (C and D) Western blotting and reverse transcription-quantitative PCR analysis of Zeb1, E-cad, N-cad, VEGF, TRAIL-R2, TRAIL-R3, Akt, Akt^p-Ser473, Akt^p-Tyr308, cyclin D1 and Bcl-2 expression in HTR-8/sv neo cells in the Zeb1, Zeb1 + Akt kinase inhibitor and vector groups **P<0.05 compared with vector group; ^##P<0.05 compared with Zeb1 group. Akt^p-Ser473, phosphorylated Akt^Ser473; Akt^p-Tyr308, phosphorylated Akt^Tyr308; E-cad, E-cadherin; IGF1, insulin like growth factor 1; N-cad, N-cadherin; si, small interfering; TRAIL-R, TNF receptor superfamily member; VEGF, vascular endothelial growth factor; Zeb1, zinc finger E-box-binding homeobox 1.

Figure 5.

Zeb1 promotes cell growth and migration through the Akt pathway. (A and B) Western blotting and reverse transcription-quantitative PCR analysis of Zeb1, E-cad, N-cad, VEGF, TRAIL-R2, TRAIL-R3, Akt, Akt^p-Ser473, Akt^p-Tyr308, cyclin D1 and Bcl-2 expression in HUVECs in the si-Zeb1, si-Zeb1 + IGF1 or scrambled groups. **P<0.05 compared with scrambled group; ^##P<0.05 compared with si-Zeb1 group. (C and D) Western blotting and reverse transcription-quantitative PCR analysis of, E-cad, N-cad, VEGF, TRAIL-R2, TRAIL-R3, Akt, Akt^p-Ser473, Akt^p-Tyr308, cyclin D1 and Bcl-2 expression in HUVECs in the Zeb1, Zeb1 + Akt kinase inhibitor or vector groups **P<0.05 compared with vector group; ^##P<0.05, compared with Zeb1 group. Akt^p-Ser473, phosphorylated Akt^Ser473; Aktp^−Tyr308, phosphorylated AktTyr308; E-cad, E-cadherin; HUVECs, human umbilical vein endothelial cells; IGF1, insulin like growth factor 1; N-cad, N-cadherin; si, small interfering; TRAIL-R, TNF receptor superfamily member; VEGF, vascular endothelial growth factor; Zeb1, zinc finger E-box-binding homeobox 1.