Role Of Zinc Finger Transcription Factor ZEB1-promoted EMT Of Endometrium In Embryo Implantation And Its Mechanism

BackgroundThe first critical step of pregnancy is the process by which the embryo implants to endometrial epithelial cells, known as implantation. Implantation is a complicated and tedious process, consists of the embryo positioning, adhering to the endometrial epithelial cell (EEC) layer, and invading into endometrial stromal cells (ESC). During this process, mulberry embryo develops into blastocyst and endometrial epithelial cell differentiates into embryos-accepting intrauterine membrane. The embryo implantation would succeed only if the development of blastocyst and the differentiation of uterine endometrium are synchronous. During the Mammalian embryo implantation process, the endometrium has receptivity to the embryo. In the reception stage, endometrial epithelial cells without adhesive ability gain adhesive ability, allowing the embryos adhere to endometrium and invade into endometrial stromal cells. The endometrial epithelial cells with adhesive ability have the following features:firstly, the endometrial epithelial barrier is destructed and the structure and function of endometrial epithelial across some membrane is shifted; secondly, endometrial epithelial cells secret phase specific proteins and glycoproteins; thirdly, endometrial stromal cells are decidual due to the regulation of secretion of endometrial epithelial cells; fourthly, the extracellular matrix of endometrial cells are degradated and reshaped. It was reported that the differentiation of endometrial epithelial cells and stromal cells is regulated by steroid hormones during menstrual cycle so as to ensure that the embryonic receptivity of endometrium occurs on 7 to 11 days after ovulation. This period is called the "window period" of embryonic implantation. Hence in the early period of embryo implantation, the destruction of the endometrial cell barrier is before cell remodeling. However, neither the mechanism of remodeling nor the relation between remodeling and cell migration and differentiation is clear.Epithelial mesenchymal transition (EMT), a biological process in which epithelial cells lose cell polarity and adhesion and acquire a migratory, mesenchymal property, is essential for a variety of physiological and pathological mechanisms, such as embryogenesis, differentiation, inflammation, wound healing and so on. EMT allows cellular metastasis and invasion especially at the precancerous stage. It has been demonstrated that he EMT process of endometrial epithelial cells, which leads to cell migration, significantly impacts on the remodeling of endometrial epithelial cells with receptivity.E-cadherin and N-cadherin are members of cadherin transmembrane superfamily. They are transmembrane adhesion molecules expressed in normal epithelial cells, and play a role in the adhesion between the same type of cells and the maintenance of cell polarity. Abundant studies showed downregulation of E-cadherin and upregulation of N-cadherin in the EMT process, which is known as E/N-cadherin switch. The switch is closely related to the rearrangement of actin. For example, the generation of stress fiber and the decrease of cortical actin both control the acceleration of cell migration. The expression of E-cadherin can be suppressed by a variety of transcription factors, such as Snail/Slug, E12/E47, Twist, and ZEB family.Besides the adhesive molecules, the cytoskeleton proteins and matrix metallo proteinases account for the occurrence and regulation of EMT. Vimentin is a cytoskeleton protein and a marker of stromal cells which is not expressed in epithelial cells. Vimenten is involved in the maintenance of the morphology of the cells and organelles, and in the promotion of cell adhesion, migration, and apoptosis. Abnormal high expression of Vimentin leads to the changes of cytoskeleton proteins and results in stromal cell-like behavior of epithelial-derived cells; it enhanced ability of invasion and metastasis. In the EMT process, the expression of Vimentin is significantly increased as well as it does in metastatic tumor cells, so it has been considered to be one of the EMT molecules. However, its effects in embryo implantation still remain unclear.ZEB1, belonging to the superfamily of zinc finger protein, has two subtypes, including ZEB1 and ZEB2. ZEB1 can inhibit the transcription of E-cadherin by binding to the segment [CACCT(G)] of E2 box on the promoter of E-cadherin gene, and hence induce the EMT and enhance the ability of cell migration and invasion. ZEB1 has been demonstrated to play critical role in the development, invasion and migration of cancers. It regulates the Notch signal pathway by negative feedback regulation based on the cross inhibition with microRNA-200. In the menstrual cycle, the expression of ZEB1 at the myometrium and endometrial stromal was higher at the progestational phase with high level of estrogen than the proliferative phase with low level of estrogen. In addition, the high expression of ZEB1 was also observed at the maternal-fetal interface of fetal bovine, and EMT is found in the embryo implantation of mouse. Meanwhile, EMT occurs with embryo adherent to endometrial cells in the embryo implantation in vitro, accompanied by upregulation of vimentin and downregulation of E-cadherin and Keratin. Additionally, EMT is altered by estrogen and progestogen level.The binding of ZEB1 to E-BOX of E-cadherin induces the EMT process, consequently results in the migration and invasion of cancer cell. At present, the mechanism of the EMT still pose challenges in the research area of implantation and embryo development. The implantation of embryo is similar to the invasion of cancers, and are both paralleled with EMT process in which the transcription factor ZEB1 plays a part. ZEB1 is expressed at the myometrium and endometrial stromal, particularly high at the progestational phase than the proliferative phase. As the progestational phase is the implantation window, we assumed that ZEB1 probably participates in the EMT process and critically influences the embryo implantation. In the study of heterozygous with mouse 8EF1/ZEB1 LacZ, ZEB1 was at the myometrium with ER receptor expression and mesenchymal cells, controlled by the level of estrogen and progestogen. According to the association between the factors related to embryo implantation such as E-cadherin, estrogen and progestogen, and ZEB1, we speculate that ZEB1 is involved in the embryo implantation through promotion of the EMT of endometrial cells at the implantation period, and this process is regulated by estrogen and progestogen.A number of studies revealed the role of ZEB1 in the migration and invasion of cancer. Furthermore, it has been demonstrated that, ZEB1 acts as a marker of migration in mesoderm and neural crest cells, and as an important controller of EMT. Previous studies indicated an important role of ZEB1 in embryo implantation. However, the related functional study and its mechanism in the embryo implantation are still in the blank. In clinic, the cause of recurrent miscarriage, repeated failure of embryo fertilization, and unexplained infertility, is possibly due to failure of the embryo implantation. Therefore, it is urgent to study the cause and the mechanisms of failure of embryo implantation.ObjectivesBy using human in vitro embryo implantation model with ZEB1 shRNA lentivirus vector with RNA interference technology, and in vivo pregnant mouse model, the present study aimed to evaluate the effects of ZEB1 in embryo implantation, and to investigate the mechanisms of ZEB1 on mediating EMT process, in order to obtain theoretical foundation for the treatment of assisted reproductive and infertility.Materials and methods1. QRT-PCR, immunohistochemistry and Western blot was employed to assess the expression pattern of ZEB1 in endometrium at the phase of menstrual cycle of normal human.2. ZEB1 shRNA lentivirus vector was constructed by using RNA interference technology. And human endometrium carcinoma cell line RL95-2 was transfected to establish stable cell lines. QRT-PCR and Western blot was used to examine the transfection efficiency. Wound healing assay, EDU proliferation assay and DNA replication experiments were performed. The in vitro embryo implantation model was established based on endometrium carcinoma cell line RL95-2 and human choriocarcinoma cell line JAR, on which we studied the function of ZEB1 in human embryo implantation, and explored whether ZEB1 led to EMT of endometrium epithelial cells and promotion of embryo implantation through downregulating E-cadherin and upregulating Vimentin.3. The pregnant mouse model was established, on which the expression pattern of ZEB1, E-cadherin and Vimentin was tested at the pre-implantation, implantation and post-implantation stage of mouse, with emphasis on the role of ZEB1 in the EMT process of mouse embryo implantation.4. The antisense oligonucleotide of ZEB1 was injected into the uterine of mouse at the pre-implantation stage, and the pregnancy outcome of mouse was observed for preliminary discussion of the effects of ZEB1 on mouse embryo implantation.5. Statistical Methods:SPSS 20.0 statistical software was employed. X2 test was used for analysis of count data. Measurement data were expressed as mean ± standard deviation (x±S), while t test and F test were used. The comparison of two data sets was done by t test, and more than two data sets were analyzed by using analysis of variance. Homogeneity of variance was analyzed, and F test was used for multiple samples with omogeneity of variance and Wechl test was used for multiple samples with no homogeneity of variance. P<0.05 was considered statistically significant.Results1. Q-PCR and western blot results showed that, ZEB1mRNA and protein was expressed in human endometrial at prophase(mRNA:0.018±0.006; Protein: 1.000±0.100), metaphase(mRNA:0.175±0.006; Protein:0.987±0.076) and advanced stage(mRNA:0.019±0.004; Protein:1.170±0.176) of the proliferative period, in particular more significantly in the mid-secretory phase(mRNA:0.043±0.015; Protein: 1.853±0.150) than other two phases(Pro:0.022±0.007; Protein:1.357±0.832)(Met: mRNA:0.026±0.007; Protein:1.330±0.128), and there are significant difference (mRNA:F=12.99, P=0.000;Protein:F=19.988, P=0.000).2. ZEB1-shRNA lentivirus vector and negative control of ZEB1-shCtrl were constructed successfully and were transfected to RL95-2 cells, so that the stable ZEB1-shRNA-RL95-2 cell lines ZEB1-shCtrl-RL95-2 cell lines were obtained. Functional assay revealed that the expression of ZEB1(0.326±0.053 vs 1.001± 0.138, t=-12.525, P=0.000) and vimentin (0.498±0.117 vs 1.000±0.230, t=-4.403, P=0.000) was downregulated, while that of E-cadherin (3.260±0.342 vs 1.001± 0.138, t=13.869, P=0.000) was upregulated. Meanwhile, the proliferation (0.72±0.07 vs 0.94±0.09, F=--4.329, P=0.003) and migration of cells, DNA replication capability (43.26±5.11 vs 62.73±7.29, F=3.788,0.019)of 72h and adhesion of JAR sphere (0.398±0.065 vs 0.833±0.053, t=16.425, P=0.000) were significantly reduced compared to ZEB1-shCtrl-RL95-2 cell, and there was statistically significant between two groups.3. The mRNA and protein expressions of ZEB1 in the endometrium of nonpregnant and 2.5-6.5 days pregnant mouse was detected by QRT-PCR and Western blot, respectively, and the results showed that ZEB1 mRNA and protein expressions in the endometrium of pregnant (mRNA:D2.5:0.028±0.007, D3.5: 0.031±0.007, D4.5:0.042±0.006, D5.5:0.031±0.008, D6.5:0.025± 0.007)(Protein:D2.5:0.032±0.005, D3.5:0.035±0.011, D4.5:0.041±0.002, D5.5: 0.029±0.012, D6.5:0.028±0.005) mouse were significantly higher than that in nonpregnant (mRNA:0.018±0.009, Protein:0.027±0.005) mouse, depending on the duration of pregnancy. The expression reached the peak at the implantation window D4.5, with significant difference between each pregnant groups (mRNA: F=11.165, P=0.000; Protein:F=14.66, P=0.000), followed by a decrease at the D5.5. Immunohistochemistry results demonstrated that, ZEB1 protein was expressed in endometrial tissue of both nonpregnant and pregnant mice. The main part of positive staining was located in the nuclei, particularly in the epithelial cells (including luminal epithelium and glandular epithelium). ZEB1 expression was abundant in the nuclei in stromal cells of pregnant mice. The positive ZEB1 protein expression showed a gradual increasing trend in mouse endometrial luminal epithelium and glandular epithelium, reached a peak at D4.5, and turned weak at D5.5. Whereas in mouse stromal cells, it could hardly be observed at D2.5, emerged at D3.5, and decreased from D6.5.4. The mRNA and protein expression of E-Cadherin in endometrium was higher in non-pregnant(mRNA:1.87±0.66; Protein:1.000±0.251) mouse endometrium than that in pregnant (mRNA:D2.5:1.10±0.389, D3.5:0.75±0.15, D4.5:0.30±0.13, D5.5:0.39±0.11, D6.5:0.80±0.22)(Protein:D2.5:0.633±0.119, D3.5:0.553± 0.058, D4.5:0.213±0.029, D5.5:0.517±0.086, D6.5:0.520±0.026) mice during early pregnancy. Each pregnant group showed significant difference (mRNA: F=26.379, P=0.000); Protein:F=32.188, P=0.001). The expression kept gradual decreasing along the time, and reached to bottom at D4.5, and began to increase at D6.5. The main site of E-cadherin positive staining was located in the cytoplasm, particularly in epithelial cells (including luminal epithelium and glandular epithelium), and showed gradual decreasing with its bottom at D4.5, but no expression was observed in the stromal cells in pregnant mouse.5. The mRNA and protein expression of Vimentin in endometrium of non-pregnant (mRNA:0.59±0.21; protein:0.99±0.40) mouse was lower than that in early pregnant (mRNA:D2.5:1.19±0.15, D3.5:1.59±0.40, D4.5:2.92±0.99, D5.5:2.13 ±0.51, D6.5:1.82±0.59)(Protein:D2.5:1.23±0.24, D3.5:1.64±0.26, D4.5:2.37 ±0.20, D5.5:1.67±0.11, D6.5:1.52±0.31) mice, with significant difference between each group (mRNA:F=23.914, P=0.000; Protein:F=9.191, P=0.001). It showed a gradual increasing trend day by day, reached to a peak at D4.5, and started to decline at D5.5.6. At one day before pre-implantation of embryos, with injection of ZEB1-shRNA dilution or ZEB1-shCtrl negative control into the uterine horn, the mice were fed continually for 8 days, and were sacrificed by cervical dislocation. The effects of ZEB1-shRNA on embryo implantation rate were evaluated. The embryo implantation rate in ZEB1-shRNA group was significantly lower than the saline side of the uterus (1.875±0.835 vs 7.375±1.408, t=9.505, P=0.000). Whereas the embryo implantation rate inZEB1-shCtrl group did not differ from the saline side of the uterine (6.375±1.119 vs 7.500±1.119, t=1.888, P=0.080).Conclusions1. ZEB1 was expressed in the uterine endometrial during the menstrual cycle, with especially high level at mid-secretory phase. Furthermore, its expression level was high in human implantation window, suggesting that ZEB1 may be involved in the regulation of endometrial receptivity and played an important role in early implantation of human embryo.2. ZEB1 knockout markedly suppressed cell proliferation, migration in RL95-2 and adhesion rate of JAR sphere in model embryo, paralleled with increased expression of E-cadherin and reduced expression of vimentin, indicating that ZEB1 possibly promoted the process of the EMT process during embryo implantation through downregulating E-cadherin and upregulating vimentin, resulting a transformation of endometrial epithelial cells to mesenchymal cells, and thus the cell migration capability was increased. Migration of epithelial cells led to damage of epithelial cell barrier, and finally altered endometrial receptivity to promote adhesion and implantation of embryos.3. High expression of EMT marker factor ZEB1 and vimentin and low expression of E-cadherin in mouse endometrium at the implantation window suggested that the EMT process occurs in mouse embryo implantation process. ZEB1 may also induces the EMT by downregulating E- cadherin, triggering cadherin switch, upregulating vimentin and inhibiting the expression of adhesion molecules, and consequently causing changes of cytoskeletal and stress fibers, which is favorable for endometrial epithelial receptivity. Thus the epithelial barrier of implantation site was damaged and remodeled, promoting the probability of implantation and decidualization change of endometrium.4. Injection of ZEB1 antisense oligonucleotides into the endometrium of pre-implantation mouse reduced the implantation rate, demonstrating that ZEB1 plays an important regulatory role in embryo implantation, probably due to promoting the EMT process and the differentiation and remodeling of endometrial epithelial cell in endometrial epithelial cells.