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The role of Zeb1 in the pathogenesis of morbidly adherent placenta

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.


Introduction
Morbidly adherent placenta (MAP) refers to abnormal attachment of the placenta to the uterus. This includes abnormal placenta attachment depth, including placenta accreta (PA), but not placenta previa (PP), which is a condition caused by a low lying placenta (1). PA refers to a pathological condition where abnormal placental villi implant into the uterine wall, due to the partial or total absence of the decidual basal layer. Placental villi may not only invade the uterine muscular layer, but may also penetrate the uterine wall into the outer serosa layer, extending even to the bladder, making dissection of the placenta from the uterus impossible (2). A previous study reported that the incidence rate of PA has increased in the past 50 years (3). For patients with PA, dissection of the entire placenta during delivery is difficult, which may lead to intrapartum and postpartum hemorrhage. The average hemorrhage size is large, and it is the main cause of perinatal emergency hysterectomy (4). The maternal mortality rate associated with PA is a serious obstetric complication (5,6). It is necessary to identify key proteins and pathways affecting placental implantation for further prediction and treatment.
Zinc finger E-box-binding protein (ZEB) serves an important role in cell differentiation and embryonic development (7). It has been reported that ZEB is associated with biological processes, including tumor invasion and metastasis (8). Although embryo implantation into the endometrium is similar to the invasion mechanism of tumors, limited research has been conducted on ZEB in pernicious PP (9). Zeb1 is a nuclear transcription factor of zinc finger proteins. It can bind to the E2 box on the promoter region of the E-cadherin-coding gene, thus inhibiting E-cadherin transcription, inducing epithelial-mesenchymal transition (EMT), and enhancing cell invasion and metastasis; therefore, it may serve an important role in cell differentiation and embryonic development, which are important developmental processes (10,11). There is also the process of EMT during embryo implantation and development. Cell invasion of the endometrial layer is an important factor that determines the success of embryo implantation. Previous studies have reported that the expression of EMT markers, including Zeb1, Zeb2, Twist family BHLH transcription factor and Snail family transcriptional repressor 1 in the outer cell layer of trophoblasts is increased at 22 days gestation (12). Therefore, it may be speculated that Zeb1 is involved in regulation of trophoblastic invasion (13). However, to the best of our knowledge, research into human chorionic villi is limited.
The present study used immunohistochemical methods to detect the protein expression levels of Zeb1 in chorionic tissues from 60 patients, including 20 patients with pernicious PP and PA, 20 patients without PA (UPA) and 20 patients in late pregnancy that delivered by cesarean section, in order to examine the association between Zeb1 and PA, and to analyze the effects of Zeb1 on PA.

Materials and methods
Sample collection. The placental samples were obtained from patients at the Shengjing Hospital of China Medical University between January 2015 and January 2017 (Table I). Among the patients, 20 had pernicious PP with PA (PA group). The PA group consisted of patients with abnormal placental villi invading the myometrium of the uterus, according to the International Diagnostic Criteria of PA (14). Pathological diagnosis was determined by observation of placental villi having directly invaded the uterine myometrium on the excised uterine specimens. The average age of the PA group was 27.38±3.98 years. The UPA group comprised 20 patients without PA. The UPA group consisted of patients in which invasion of abnormal placental villi into the myometrium of the uterus was not observed, according to the International Diagnostic Criteria of PA. The average age of the UPA group was 28.12±2.56 years. Furthermore, 20 placental samples were obtained from patients in late pregnancy that delivered by cesarean section (normal group); their average age was 26.99±3. 31.
The selection criteria were as follows: i) Patients had not entered labor; and ii) had not had a history of cesarean section. The exclusion criteria for the three groups were as follows: i) History of hypertension; ii) history of nephropathy and diabetes; iii) history of anemia or other blood diseases; iv) the current pregnancy was via vaginal delivery; v) fetal distress or intrauterine growth retardation; and vi) a history of blood transfusion and immunotherapy.
Tissue samples were obtained from patients undergoing surgery at Shengjing Hospital of China Medical University. Part of the excised tissue was fixed in 10% buffered formalin (4˚C, overnight) and part of the sample was snap-frozen at -80˚C for western blotting. Clinical samples were collected after written informed consent was obtained from patients, and the study was approved by the Ethics Committee at the Academic Medical Center of Shengjing Hospital of China Medical University.
Immunohistochemical evaluation. Tissue sections were immunohistochemically stained for Zeb1, vascular endothelial growth factor (VEGF) and E-cadherin. Samples were fixed in 10% buffered formalin (4˚C, overnight). Serial 5 µm sections were cut using a microtome and affixed onto positively charged slides. Tissues were deparaffinized and rehydrated through graded xylene and in descending alcohol series. The paraffin sections were subsequently de-waxed in water and incubated with 3% H 2 o 2 for 10 min at room temperature. The sections were incubated with 3% goat serum (cat. no. 16210064; Thermo Fisher Scientific, Inc.) at room temperature for 10 min. Immunohistochemistry (IHC) was conducted using rabbit primary antibodies against Zeb1 (1:800; sc-515797), VEGF (1:800; sc-53462) and E-cadherin (1:500; sc-71007) (Santa Cruz Biotechnology, Inc.) at 4˚C overnight. An avidin-biotin-horseradish peroxidase complex immunodetection kit (D110073-0500; Shanghai Shenggong Biology Engineering Technology Services, Ltd.) was used to detect the reaction according to the manufacturer's instructions. Finally, the slides were dried, mounted with Canada balsam, covered and were examined using a light microscope (magnifications, x100 and x400). Staining intensity was scored on a scale of 0-4, as follows: 0, no staining; 1, weak staining in 5-25% of areas; 2, weak staining in 25-50% of areas; 3, moderate staining in 51-75% of areas and 4, strong staining in >75% of areas.
Cell culture. HTR-8/sv neo cells and human umbilical vein endothelial cells (HUVECs) were supplied by the China Center for Type Culture Collection. The cells were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C in an atmosphere containing 5% CO 2 .
Cell viability assay. The MTT assay was employed to assess cell viability. HTR-8/sv neo cells and HUVECs were cultured in 96-well plates at a concentration of 1x10 4 cells/ml and were transfected with a Zeb1 vector or si-Zeb1 for 12, 24, 36 or 48 h. A total of 0.01 ml MTT solution (5 mg/ml) was added to each well. After 4 h of incubation at 37˚C, the medium was replaced with 0.2 ml DMSO for 15 min. Subsequently, optical density was measured at a wavelength of 490 nm.
Cell cycle assay. In total, 1x10 6 HTR-8/sv neo cells or HUVECs were seeded into 6-well plates and transfected with a Zeb1 vector or si-Zeb1 for 24 h. Cells were fixed in 75% ethanol at 4˚C overnight. Following staining using 10 µg/ml propidium iodide (PI) and 100 µl RNase A (100 µg/ml; Beyotime Institute of Biotechnology) for 20 min at room temperature, cell cycle progression was analyzed by flow cytometry within 1 h. The cell cycle was then analyzed using a flow cytometer (BD FACSCalibur; BD Biosciences) and FlowJo V10 (FlowJo, LLC).
Apoptosis assay. In total, 1x10 6 HTR-8/sv neo cells or HUVECs were seeded into 6-well plates and transfected with a Zeb1 vector or si-Zeb1 for 24 h. Cells were washed twice with cold PBS, and stained with 5 µl Annexin V-fluorescein isothiocyanate (FITC)/10 µl PI which were included in the Annexin V-FITC/PI apoptosis kit (Beyotime Institute of Biotechnology). Cells were incubated for 15 min at room temperature. Subsequently, 400 µl binding buffer (Beyotime Institute of Biotechnology) was added to each tube and the apoptosis rate was measured by flow cytometry within 1 h. Apoptosis was then detected using a flow cytometer and analyzed using FlowJo.
Transwell migration assay. In total, 1x10 5 HTR-8/sv neo cells or HUVECs in 200 µl DMEM were transfected with Zeb1 vector or si-Zeb1 for 24 h in 100 µl FBS-free DMEM. Cells were then placed in the upper chamber of a Transwell system, whereas the lower compartment was filled with 600 µl DMEM containing 10% FBS. After 8 h of incubation at 37˚C, cells were fixed in 75% ethanol at 4˚C overnight. The cells that had migrated to the lower surface of the filter were stained with 4% trypan blue (Beyotime Institute of Biotechnology) for 20 min at room temperature and counted under a light microscope (magnification, x400).

Reverse transcription-quantitative PCR (RT-qPCR).
Total RNA was extracted from HTR-8/sv neo cells, HUVECs or tissues using the RNA Isolation kit (Tiangen Biotech Co., Ltd.), according to the manufacturer's protocol, and samples were stored at -80˚C until subsequent use. The RNA Reverse Transcription kit (Beyotime Institute of Biotechnology) was used for the RT of RNA into cDNA using the following conditions: 37˚C for 15 min, and followed by 85˚C for 5 sec. RT-qPCR was subsequently performed using a SYBR ® Green Real-time PCR Master Mix (Toyobo Co., Ltd.). RT-qPCR was performed using MX3000P Real-time PCR instrument (Agilent Technologies, Inc.), according to the manufacturer's protocol, using the following conditions: 94˚C for 5 min, and followed by 30 cycles of 94˚C for 30 sec, 58-61˚C for 30 sec. Primer sequences for detection of mRNA expression are shown in Table II. The relative gene expression levels were calculated using the 2 -ΔΔCq method (15). All experiments were performed in triplicate.
Western blot analysis. Tissues, HTR-8/sv neo cells and HUVECs were lysed in lysis buffer [25 mM Tris (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA]. The cell lysates were collected by centrifugation at 12,000 x g for 10 min at 4˚C. Total protein concentration was quantified using a bicinchoninic acid protein assay kit (Applygen Technologies, Inc.), and western blot analysis was performed. A total of 30 µg protein were separated by 10% SDS-PAGE and were transferred to a polyvinylidene difluoride membrane (Shanghai Shenggong Biology Engineering Technology Service, Ltd.). Following blocking with 3% BSA in TBS containing 0.1% Tween-20 for 3 h at room temperature, the membranes were incubated overnight at 4˚C with primary antibodies. The following primary antibodies were used: Zeb1 After several washes, the membranes were incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (goat anti-mouse; 1:5,000; sc-2031; goat anti-rabbit; 1:5,000; sc-2030; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Proteins bands were visualized using enhanced chemiluminescence kits (GE Healthcare). After Akt/E-cadherin/TRAIL-R2 antibodies were detected, the membrane was stripped for 30 min at 55˚C with a wash solution (50 mM Tris, 2% SDS, 100 mM 2-mercaptoethanol), washed three times with TBS-10% Tween-20, re-blocked and incubated with Akt p-Ser473 /pAkt p-Tyr308 /N-cadherin/TRAIL-R3/GAPDH antibodies. All reactions were repeated at least in triplicate.
Statistical analysis. All reactions were repeated a minimum of three times. All data are presented as the mean ± standard deviation. Statistical analysis (two-tailed) of more than two groups was performed by one-way analysis of variance, followed by Tukey's post-hoc test for multiple comparisons. A Student's t-test was used to compare differences between two groups. Statistical analyses were conducted using GraphPad Prism 6 for Windows (version 6.05; GraphPad Software, Inc.) P<0.05 was considered to indicate a statistically significant difference.

Results
Zeb1 expression in placenta tissues. The expression of Zeb1 in placenta tissues was assessed by IHC (Fig. 1A). The results demonstrated that Zeb1 expression was highest in the PA group, and was higher in the UPA group compared with in the normal group. In addition, IHC revealed that VEGF expression was higher, whereas E-cadherin expression was lower in the PA group compared with in the normal group ( Fig. 1B and C). Western blot analysis and RT-qPCR demonstrated that the expression levels of Zeb1, N-cadherin, VEGF and TRAIL-R3 (P<0.05) were higher in the PA group compared with in the control group, whereas E-cadherin and TRAIL-R2 expression were lower (P<0.05; Fig. 1D and E)

Si-Zeb1 inhibits the viability and migration of HTR-8/sv neo cells.
The MTT assay revealed that Zeb1-silencing [depletion was confirmed using western blotting and RT-qPCR ( Fig. 2E and F)] inhibited the viability of HTR-8/sv neo cells (P<0.05; Fig. 2A). Flow cytometry was used to evaluate cell cycle progression of HTR-8/sv neo cells previously exposed to si-Zeb1 for 24 h. si-Zeb1 induced a decrease in the percentage of cells in S phase, and increased the number of cells in G 1 phase, compared with in the control group (P<0.05; Fig. 2B). In order to assess whether Zeb1 can inhibit apoptosis, flow cytometry was used to assess HTR-8/sv neo cells. The results demonstrated there was an increase in si-Zeb1-transfected apoptotic cells (P<0.05; Fig. 2C). The effects of Zeb1 on HTR-8/sv neo cell migration were assessed using Transwell assays. The results showed that si-Zeb1 significantly inhibited migration of HTR-8/sv neo cells (P<0.05; Fig. 2D). Western blot analysis and RT-qPCR revealed that transfection with si-Zeb1 for 24 h decreased the expression levels of N-cadherin, VEGF, TRAIL-R3, cyclin D1 and Bcl-2 (P<0.05), and promoted the expression levels of E-cadherin and TRAIL-R2 in HTR-8/sv neo cells (P<0.05; Fig. 2E and F). HUVEC viability and migration are inhibited by si-Zeb1. The results demonstrated that si-Zeb1 [depletion was confirmed using western blotting and RT-qPCR ( Fig. 3E and F)] inhibited the viability of HUVECs by inhibiting G 1 /S transformation and promoting apoptosis (P<0.05; Fig. 3A-C). The results of a Transwell assay revealed that si-Zeb1 could significantly inhibit the migration of HUVECs (Fig. 3D). Western blot analysis and RT-qPCR showed that si-Zeb1 could inhibit the Zeb1 promotes cell growth and migration through the Akt pathway. Since Zeb1 is involved in regulation of the Akt pathway (19), this study examined whether the role of Zeb1 in PA was partly mediated by the Akt pathway. To achieve this, si-Zeb1 cells were treated with an Akt activator and Zeb1 overexpressing cells were treated with an Akt inhibitor. Western blot analysis and RT-qPCR revealed that IGF1 reversed the effects of si-Zeb1 on Akt p-Ser473 , Akt p-Tyr308 , N-cadherin, VEGF, TRAIL-R3, cyclin D1, Bcl-2, E-cadherin and TRAIL-R2 in HTR-8/sv neo cells (P<0.05; Fig. 4A-D). Similar results were observed in HUVECs, as determined by western blotting and RT-qPCR (P<0.05; Fig. 5A-D).

Discussion
PA is a complex process, the underlying mechanism of which remains unclear. The balance between normal placental villus infiltration and decidual tissue reaction is the basis for a normal pregnancy and a smooth delivery (1). It has been suggested that decidual defects, excessive invasion of trophoblasts and abnormal neovascularization of the uterus and placenta can lead to PA (20). When PA occurs, placental villi adhere tightly to the basal plate of the decidua basalis and invade the myometrium of the uterus, indicating that the migratory and invasive ability of trophoblasts outside villi is enhanced (1,21). In the present study, it was demonstrated that the expression of Zeb1 was significantly higher in PA tissues compared with in the control and UPA groups. Zeb1 can promote the proliferation and migration of trophoblasts and umbilical vein endothelial 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.
cells (22). Therefore, it may be concluded that Zeb1 serves an important role in PA. PA is a process of tissue migration, in which EMT and mesenchymal-epithelial transition (MET) serve important roles, and the migratory ability of cells is greatly improved (23). Zeb1 may serve an important role in placental implantation as a regulator of EMT. The process of EMT involves several changes in protein expression (24). It has previously been reported that Zeb1 can regulate E-cadherin gene expression to participate in the regulation of EMT (7). When PA occurs, the invasive ability of trophoblasts is increased and the MET process cannot be completed, which is manifested by an increase in the number and volume of invasive trophoblasts during PA (9). In the present study, the expression levels of Zeb1 and E-cad were detected in the three patient groups: PA, UPA and normal. The results revealed that the expression levels of Zeb1 were significantly upregulated, whereas the expression levels of E-cadherin were downregulated in the PA group. These results are consistent with previous studies (25,26), which reported that Zeb1 and E-cadherin can participate in the occurrence and development of placental implantation.
It has been suggested Zeb1 can induce angiogenesis and promote the expression of VEGF, thereby promoting angiogenesis (27). VEGF is involved in the occurrence of preeclampsia. PA is also associated with vascular abnormalities, and the process of PA can be regulated by Zeb1 (28). In this study, it 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. was indicated that the expression levels of Zeb1 and VEGF were significantly upregulated in the PA group, thus indicating that Zeb1 and VEGF may promote PA.
The Akt signaling pathway serves an important role in embryogenesis (29). Overactivation of the Akt signaling pathway has been shown to be associated with macrosomia and embryonic development (30). In addition, overactivation of the Akt signaling pathway can induce cell overproliferation and enhance migratory abilities, which has been widely demonstrated in various cells (31). EMT is the basic process of embryonic development, tissue remodeling and wound healing (30). In recent years, EMT has gained increasing attention in cancer progression, invasiveness and metastasis (31). Through the EMT process, cells may acquire an invasive phenotype, which may contribute to tissue invasion (32). Zeb1 has been reported to activate a variety of downstream pathways, including PI3K, Smads and MAPK, which are involved in Zeb1-induced EMT (33). In this study, the expression of Zeb1, Akt, Akt p-Ser473 , Akt p-Tyr308 , E-cadherin, N-cadherin, VEGF, TRAIL-R2, TRAIL-R3, cyclin D1 and Bcl-2 were detected following the addition of the Akt activator IGF1 to Zeb1-silenced cells. The results indicated that Zeb1 silencing could decrease the phosphorylation levels of Akt without affecting the total protein levels of Akt, whereas IGF1 could counteract the change in protein expression induced by Zeb1 silencing. In addition, treatment with an Akt kinase inhibitor weakened the effects of Zeb1 overexpression on protein production to some extent. These findings suggested that the regulatory effect of Zeb1 on cellular biological function may be achieved through the Akt signaling pathway to some extent.
In the present study, the sample size was limited, and other possible signaling pathways that Zeb1 may regulate were not screened; therefore, further examination is required. In addition, there are further limitations to this study. Notably, SC79, which is more specific than IGF-1, was not used as an Akt activator. Therefore, in future studies, a more specific activator of Akt should be used. Furthermore, IGF-1 can produce off-target effects that do not involve Akt. Further analysis should be conducted in a follow-up study.
In conclusion, this study demonstrated that Zeb1 may promote placental implantation by activating the Akt signaling pathway. The present findings support the potential of Zeb1 as a novel diagnostic and treatment target for PA.