Potential role of FoxO3a in the regulation of trophoblast development and pregnancy complications

Abstract The forkhead box O3a protein (FoxO3a) has been reported to regulate tumour invasion and migration, but little is known about the molecular mechanism or its role in trophoblast invasion and migration into the uterus. In this study, we aim to explore its role in trophoblast development and placenta‐related pregnancy complications and the potential mechanism. Levels of FoxO3a and its phosphorylated form (p‐FoxO3a) in placental tissue from healthy pregnant women and pre‐eclampsia patients were first compared. Then, HTR‐8/SVneo cells were transfected with lentiviral vectors to deplete and overexpress FoxO3a. Western blot, immunohistochemistry, Cell Counting Kit‐8, wound‐healing assay, Matrigel invasion assay, cell apoptosis, cell cycle assay, RNA sequencing, qRT‐PCR and ChIP‐qPCR were performed on the cells to study the potential role of FoxO3a and the underlying mechanism. We found the expression of FoxO3a was decreased, whereas p‐FoxO3a was increased in pre‐eclampsia placentae. FoxO3a depletion significantly reduced transcription of the promoter region of intercellular cell adhesion molecule‐1 (ICAM1) gene in ChIP assays and led to reduced invasion and migration of trophoblast cells, arrested cell cycle in G1 phase and increased apoptosis under oxidative stress. Our results suggested that FoxO3a may play a role in the regulation of trophoblast invasion and migration during placental development, which may be because of its affinity to the ICAM1 promotor.


| INTRODUC TI ON
The placenta is the intermediary organ that connects the mother and foetus during pregnancy. Its development started with trophoblast invasion and migration into the uterus. [1][2][3] Dysfunction of trophoblast cell results in superficial invasion and insufficient remodelling of spiral uterine artery, which ultimately leads to placental dysplasia and pregnancy complications including pre-eclampsia, foetal growth restriction, still birth and preterm birth. [4][5][6][7] Therefore, the invasion and migration capacity of trophoblast is worthy of attention when investigating the aetiology of the placenta-related pregnancy complications.
The invasion, migration, apoptosis and differentiation of trophoblast cells are regulated by different transcription factors and signalling pathways. [8][9][10][11] The forkhead box O3a (FoxO3a) is an important member of the O subfamily of the forkhead protein transcription factor family (Foxes). 12 It is expressed in various organs and tissues such as placenta, heart, vascular endothelium and fat and is involved in biological processes such as cell development, metabolism, apoptosis, autophagy and anti-oxidative stress. [13][14][15][16] The function of FoxO3a in tumour cells has been studied previously. When the phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) are activated, FoxO3a is phosphorylated and transferred from the nucleus to the cytoplasm, thus reducing the FoxO3a level in the nucleus and the expression of downstream genes promoted by FoxO3a. 17,18 The decreased FoxO3a expression in tumour cells was reported to be associated with decreased MMP1, MMP9 and MMP13 levels and inhibited invasion and migration. [19][20][21] In another study, FoxO3a activated by PI3K or AKT inhibitors acted like a metastasis inducer. 22 However, the molecular mechanism of how FoxO3a promotes the expression of downstream genes has not been clarified yet. Besides, the behaviour of trophoblasts is somewhat similar to tumour cells, 23 in that both types of cells invade to adjacent tissue, with accelerated metabolism and cell cycle. Although studies have shown that FoxO3a plays an important regulatory role in tumours, its effects on trophoblast migration, invasion, metabolism, cell cycle and apoptosis are unknown.
Hence, this study aims to investigate FoxO3a in HTR8/neo cell   line (a well-recognized cell line similar to human trophoblast cells) and the related mechanism, with the hope of understanding its role and related mechanism in placental development and placentarelated pregnancy complications. pre-eclampsia (PE, n = 7) and age-matched normotensive pregnancies (NC, n = 7) underwent elective caesarean section at the Department of Obstetrics were randomly recruited into this study.

| Human placenta samples
The diagnosis of severe pre-eclampsia was based on the guidelines of the American College of Obstetrics and Gynecology (ACOG). 24 The exclusion criteria included diabetes, chronic hypertension, immune diseases, infections and pregnancy complications other than pre-eclampsia. All the women in the study had singleton pregnancy.
The clinical characteristics of the participants are shown in Table 1.
Placental tissue was collected, soaked in iced saline and quickly transferred to the laboratory for further preparation. The samples were cut into small pieces and washed with iced PBS for three times.
A proportion of the specimen were frozen at −80°C for Western blot analysis, and the remaining was embedded with paraffin for immunohistochemistry analysis.

| Western blot
Placental and cellular protein was extracted using RIPA lysis buffer

| Cell migration assay
Wound-healing assay was used to evaluate the migration of HTR8-S/ Vneo cells. 5 × 10 5 cells were seeded into each well of a 6-well plate.
When the cells reached 90% confluence, the cells were scratched with a 200 µL pipette tip and washed with PBS. Images were taken at 0h and 24h after the scratch. The wound-healing rate was measured with ImageJ software. Each experiment was performed in triplicate.

| Cell invasion assay
The invasiveness of the HTR8/SVneo cells were detected using Matrigel invasion assay. Four hours before seeding the cells, The invasion rate was measured with ImageJ software. Each experiment was performed in triplicate.

| Cell apoptosis and cell cycle assay
The three groups of HTR8/SVneo cells, treated with or without SNP, were collected in the logarithmic growth phase. Cells digested with trypsin were washed twice with PBS and suspended in 500 μL PBS for apoptosis assay, or in 100 μL PBS followed by addition of 500 μL iced 75% ethanol for cell cycle assay. A flow cytometer (CytoFLEX, eBioscience) was used to analyse the samples for apoptosis and cell cycle according to the manufacturer's instructions.

| Lower expression of FoxO3a was found in preeclampsia placenta
The expression of FoxO3a in the placenta of pregnant women with pre-eclampsia was significantly lower than those with normal pregnancy, whereas the expression of phosphorylated FoxO3a was remarkably higher in pre-eclampsia ( Figure 1A).
The immunohistochemistry results revealed that FoxO3a was present in trophoblast, which also expresses CK7 and HLA-G ( Figure 1B).

| FoxO3a was knocked down and overexpressed in HTR8-S/Vneo cells
To explore the role of FoxO3a in pre-eclampsia, the FoxO3a gene was overexpressed or knocked down through lentiviral transfection.

| FoxO3a regulates ICAM1 transcription
To explore the molecular mechanism related to FoxO3a, we performed RNA-seq test and discovered 469 differential genes in the shFoxO3a group, including 321 up-regulated ones and 148 downregulated ones ( Figure 3A). Disease ontology analysis revealed that 28 out of these 469 genes were related to pre-eclampsia ( Figure 3B).
Gene ontology analysis of the 469 genes showed that cell migration was the most affected biological process ( Figure 3C). KEGG pathway analysis unveiled that FoxO3a was related to a number of signalling pathways, such as TNF, NF-kappa B, IL-17 and AGE-RAGE ( Figure 3D). Protein-protein interaction (PPI) analysis found that FoxO3a was directly related to genes downstream of the TNF signalling pathway ( Figure 3E). Gene set enrichment analysis confirmed that TNF signalling pathway was down-regulated after FoxO3a depletion ( Figure 3E). qPCR unveiled that the expression of downstream genes of TNF signalling pathway, including intercellular cell adhesion molecule-1 (ICAM1), vascular cell adhesion molecule (VCAM1), prostaglandin-endoperoxide synthase 2 (PTGS2), MMP9 and MMP1, was decreased ( Figure 3F).
Notably, we found that the transcription of ICAM1 was significantly decreased (P < .001) through RNA-seq data mining. To verify the finding, we further performed ChIP-PCR and ChIP-qPCR assay on the promoter region of ICAM1 and found the enrichment of Foxo3a at the ICAM1 promoter was significantly reduced after FoxO3a depletion ( Figure 3G).

| FoxO3a knockdown and overexpression affected apoptosis, cell cycle and proliferation of HTR8/SVneo cells
The  group and oeFoxO3a group, and the latter was more remarkable ( Figure 4C).

| FoxO3a knockdown restrained the migration and invasion of HTR8/SVneo cells
We used Wound-healing assay and Matrigel cell invasion assay to investigate the effect of FoxO3a knockdown on HTR8/SVneo cells.
It was found that the wound-healing rate ( Figure 5A) and Matrigel cell invasion ( Figure 5B) of the shFoxO3a group were significantly decreased compared with the shNC group.   Figure 3G). These results were consistent with previous studies showing that ICAM1 plays an antiapoptotic effect and resists Fasinduced chondrocyte apoptosis. 27 In another report, ICAM1 was shown to be involved in signalling pathways that affect cell proliferation and apoptosis, and its expression contributes to antiapoptotic effects. 28 In addition, increased expression of ICAM1 promotes tumour development by enhancing cancer cell invasion and migration ability. 31 It is known that FoxO3a plays an essential role on cell cycle. In this study (Figure 4), we also observed that higher ratios of HTR8/ SVneo cells transfected with shFoxO3a lentiviral remained in G1/ G0 phase, and cell proliferation was restrained. Also, we observed that the migration and invasion of HTR8/SVneo cells with FoxO3a knockdown were suppressed ( Figure 5), whereas apoptosis increased ( Figure 4A). Interestingly, the apoptosis rate of cells with overexpression of FoxO3a was significantly increased. These results may be attributed to the genes downstream of FoxO3a and related to apoptosis, including Bim, Fasl, TRAIL and Bcl. [38][39][40][41][42] In conclusion, our results found that FoxO3a plays an important role in the apoptosis, migration and invasion of HTR8-S/Vneo cells, which may be partially explained by its affinity to the ICAM1 promotor.

CO N FLI C T O F I NTE R E S T S
The authors indicate that there is no potential conflict of interest.  The data that support the findings of this study are available from the corresponding author upon reasonable request.