Abstract
Recurrent pregnancy loss (RPL) is a multifactorial condition with no explanation of miscarriage in approximately half of the RPL patients, consequently leaving deep physical and emotional sequels. Transcription factor 3 (TCF3 or E2A), is a unique member of the LEF/TCF family and plays an important role in embryogenesis. However, its function in RPL is poorly understood. Using real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry, we demonstrated that TCF3 was downregulated in decidual tissues from RPL patients compared with healthy control (HC). Further, TCF3 knockdown inhibited proliferation, induced G0/G1 phase arrest, and promoted migration in human endometrial stromal cells (HESCs), while overexpression of TCF3 exhibited the opposite effects. RNA-sequencing analysis combined with gene-set enrichment analysis results showed that the mitogen-activated protein kinase pathway is potentially downstream of TCF3. Knockdown of TCF3 confirmed increased p38 phosphorylation, while overexpression of TCF3 inhibited p38 phosphorylation. Furthermore, we found that TCF3 protein level was decreased in HESCs under hypoxic incubation, while hypoxia-inducible factor-1α (HIF1A) knockdown increased the expression of TCF3. TCF3 overexpression recovered the proliferation ability of HESCs inhibited by hypoxia and reversed hypoxia-induced migration. Consistently, we found that RPL patients had a significantly higher level of HIF1A in the decidual tissue than HC. Overall, this study clarifies that increased HIF1A in the decidua contributes to the occurrence of RPL through the TCF3/p38 signaling pathway.
Introduction
Approximately a quarter of pregnancies end in miscarriages, most of which are sporadic, and the loss of pregnancy usually occurs before 13 weeks of gestation. Recurrent pregnancy loss (RPL) is a debilitating disease that involves at least two consecutive miscarriages that impair the patient’s health, both physically and emotionally (Practice Commitee of the American Society for Reproductive Medicine 2012). To date, approximately 50% of RPL cases have no clearly defined etiology (Practice Commitee of the American Society for Reproductive Medicine 2012). It has been known that chromosomal errors or reduced endometrial receptivity and selectivity of embryos may contribute to the occurrence of RPL (Teklenburg et al. 2010). Recently, it was suggested that human endometrial stromal cells (HESCs) play a critical role in RPL. In daily life, HESCs are involved in stromal and epithelial cells regeneration for endometrial repair, which is essential for embryo implantation and pregnancy sustainment (Owusu-Akyaw et al. 2019), and impaired HESCs proliferation and differentiation can lead to pregnancy failures, such as implantation failure, preeclampsia, preterm birth, and RPL (Cha et al. 2012, Aikawa et al. 2020). During the embryo implantation, HESCs move around the embryo to accommodate the trophoblast outgrowth and HESCs motility is essential for embryo implantation (Grewal et al. 2008, 2010). Weimar et al. proposed that HESCs from RPL women had an uncontrolled migratory capacity to embryos with chromosomal errors or poor developmental potential, which may be associated with the pathogenesis of RPL (Weimar et al. 2012).
The TCF/LEF family (TCFs) consists of four proteins, including TCF1, TCF3, TCF4, and LEF1 (Hrckulak et al. 2016). Among the TCFs members, TCF3 is the most abundant and unique member of the TCF/LEF family expressed in early mouse embryos (Merrill et al. 2004). TCF3 is encoded by the TCF7L1 gene and contains two alternative spliceosomes, E12 and E47, expressed through the mutually exclusive use of exons 18a and 18b (Hrckulak et al. 2016). E12 mainly exists in pluripotent cells, while E47 is highly expressed in differentiated cells (Agosto & Lynch 2018). In the early stages of embryonic development, TCF3 inhibits self-renewal and promotes the differentiation of embryonic stem cells (Martello et al. 2012). It was reported that TCF3 could act as an inhibitor of transcription independent of β-catenin (Merrill et al. 2004). Robinson et al. also found that TCFs combined with β-catenin inhibited IL-6 expression (Robinson et al. 2020). Although many studies have been conducted on TCF3, the role of TCF3 in HESCs remains to be explored.
Normally, embryos are implanted in a physiologically hypoxic environment. A lower oxygen level (5%) during human in vitro fertilization embryo culture can increase the quality of embryos and lead to a higher cumulative live birth rate than embryo culture in a normoxic environment (20%) (Van Montfoort et al. 2020). Hypoxia-inducible factor-1α (HIF1A) accumulates in response to the hypoxic environment and is rapidly degraded by the VHL-mediated ubiquitin protease system under a normoxic environment (Haase 2009). HIF1A regulates the transcription of a variety of genes that regulate angiogenesis, energy metabolism, and trophoblast differentiation (Oladipupo et al. 2011, Goda & Kanai 2012, Wakeland et al. 2017). Any abnormalities in the occurrence and duration of physiological hypoxia may be associated with pregnancy complications, such as preeclampsia and RPL (Burton & Jauniaux 2004, Sheibak 2018). However, previous studies about hypoxia and RPL mainly focused on the effect of hypoxia on extravillous trophoblasts differentiation and invasion, the effect of hypoxia on HESCs in the pathogenesis of RPL remains to be explored.
In this study, we showed that RPL patients had a lower level of TCF3 in the decidua than healthy control (HC). TCF3 knockdown inhibited HESCs proliferation and promoted HESCs migration, while TCF3 overexpression played the opposite role. Hypoxia inhibited HESCs proliferation and promoted HESCs migration by regulating the HIF1A/TCF3/p38 axis. Therefore, we propose that elevated HIF1A in the decidua is involved in the pathogenesis of RPL through inhibiting TCF3/p38 signaling axis.
Materials and methods
Patient information
A total of 18 RPL patients (mean age, 29.7 ± 6.2 years) and 20 HC women (mean age, 32.8 ± 3.1 years) from the International Peace Maternity and Child Health Hospital, China Welfare Institute, Shanghai Jiao Tong University School of Medicine were enrolled in this study between September 2019 and September 2020. All pregnancies in patients with RPL and HC were terminated at 6–11 weeks of gestation. The gestational age is 7.680 ± 0.9294 weeks in controls and 8.000 ± 1.473 weeks in RPL patients. Patients with the following diseases were excluded: (i) chromosomal disorders of the parents or abortus; (ii) endocrine disorders such as hyperthyroidism, hyperprolactinemia, and diabetes; (iii) endometritis; (iv) abnormal uterine structure or cervical incompetence; (v) other identified etiology of RPL. The HC group had successful pregnancies before and chose artificial abortions to terminate their unwanted pregnancies. This study was conducted following the guidelines of the Declaration of Helsinki. The Medical Ethics Committee of the International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine specifically approved this study. Written informed consent was obtained from all patients.
Tissue collection
All decidual tissues were collected immediately after abortion and cleaned briefly with sterile PBS (HyClone). For western blotting, part of the tissue was frozen in liquid nitrogen within 20 min of delivery and then stored at −80˚C. For real-time PCR (qRT-PCR), part of the decidual tissue was stored in RNAlater (Thermo Fisher Scientific) at −80˚C for long-term storage. For immunohistochemistry, tissues were fixed with 4% paraformaldehyde (PFA) (BBI Life Sciences, Shanghai, China) at room temperature for 24 h and then embedded in paraffin.
RNA extraction and qRT-PCR analysis
TRIzol reagent (Life Technologies) was used to extract total RNA from decidual tissues or HESCs. Two micrograms of total RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara). An SYBR Green Kit (Takara) was used for qRT-PCR. Relative mRNA expression was determined using the 2−ΔCT method, normalized against actin. The primers used were as follows: TCF3 5’-ACGAGCGTATGGGCTACCA-3’ (forward) and 5’-GTTATTGCTTGAGTGA- TCCGGG-3’ (reverse); HIF1A 5’-ATGGAGGGCGCCGGCGGCGAG-3’ (forward) and 5’-GTTAACTTGATCCAAAGCTCTGAG-3’ (reverse); Actin 5’-GTCATT- CCAAATATGAGATGCGT-3’ (forward) and 5’-GCT- ATCACCTCCCCTGTGTG-3’ (reverse).
Western blot analysis
Cells and decidual tissues were lysed with radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific) containing proteinase inhibitor cocktail (Thermo Scientific, UF286971). Proteins were separated using SDS-PAGE and transferred to 0.22-μm PVDF membranes. The membranes were blocked in 5% non-fat milk for 1 h at room temperature and then incubated with primary antibodies against TCF3 (1:2000; 67140-1-Ig, Proteintech, Wuhan, China), HIF1A (1:500; ab51608, Abcam), Phospho-p38 MAPK (1:1000; 4511, CST), p38 MAPK (1:1000; 8690, CST), and actin (1:10000, HRP-66009, Proteintech) at 4°C overnight. After rinsing three times with TBST buffer for 5 min, the membranes were incubated with secondary antibodies at room temperature for 1 h. Protein bands were visualized with an HRP chemiluminescent kit (Millipore) and quantified using Image J software (NIH). Pictures are imported into Image J and the picture mode is changed to ‘Grayscale’. The ‘rectangle’ tool from ImageJ was selected and a frame was drawn around the largest band of that row. The frame was placed on the first band and the measurement of the band was recorded. Then the frame was moved to the next lane and measurement was made for that protein for all samples (across the row).
Immunohistochemistry
A total of eight samples in the HC group and seven samples in the RPL group were collected for immunohistochemistry. Fresh decidual tissues were fixed with 4% PFA, embedded in paraffin, and serially sectioned. Immunohistochemistry (IHC) was performed according to the manual of the Rabbit-specific HRP/DAB (ABC) IHC Detection Kit (Abcam, ab64261). Antigen retrieval was performed by heating the samples in a microwave oven for 22 min in the presence of citrate antigen retrieval solution. The slides were incubated with primary antibody against TCF3 (1:1000; ab224638, Abcam) and HIF1A (1:200, ab51608, Abcam) diluted in PBS overnight at 4˚C. Control sections were run concurrently with the experimental sections using non-specific IgG. The percentage of positive cell counts was calculated using Image J (NIH). To be specific, the Cell Counter plugin was installed into the ImageJ folder. The image was opened and Plugins and Cell Counter were selected. Then the positively stained cells were clicked on directly and the results were exported into Excel for calculation.
Cell culture and hypoxic treatment
The immortalized HESCs were a kind gift from Professor Wang Haibin (Jiang et al. 2020). HESCs were cultured in Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F12, Gibco) containing 10% charcoal-stripped fetal bovine serum (CS-FBS, Biological Industries), 100 μg/mL streptomycin, 100 IU/mL penicillin, 1% insulin-transferrin-selenium solution (Thermo Fisher Scientific), and 500 ng/mL puromycin (Sigma). The cell culture medium was exchanged every 2 days. For hypoxic induction, 1 × 105 cells/ per well (six-well plate) or 2 × 105 cells/ per 6 cm dish HESCs were cultured in a hypoxic incubator (1% O2, 5% CO2, 37˚C) at indicated time points.
siRNA transfection
Three different siRNAs against TCF3 or HIF1A or randomly scrambled siRNA (negative control) were purchased form GenePharma (Shanghai) and mixed to develop a pool of siRNAs (Supplementary Table 1, see section on supplementary materials given at the end of this article). siRNAs were transfected using lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions at 50–70% confluence. Briefly, 9 µL of RNAiMAXtransfection reagent were incubated with 3 µL of siRNA for 5 min at room temperature, and the mixture was added to the cells in complete culture medium for follow-up experiments.
Cell transduction of lentivirus for TCF3 overexpression
The control vector and TCF3 overexpression lentiviruses were purchased from VectorBulider (Guangzhou). The titer of the lentivirus was 1.13 × 108 TU/mL, and the multiplicity of infection (MOI) was 20. At the confluence of 30%-50% in a six-well plate, appropriate amount of lentiviruses ware added to cells in 1 mL culture medium with 5% CS-FBS. Twelve hours later, the virus-containing culture medium was replaced with fresh complete culture medium with 10% CS-FBS. To construct TCF3 stably expressed cells, culture medium with 75 µg/mL G418 were added to cells the next day. Cells were selected with G418 for 7 days and then cultured in 10% CS-FBS medium.
Immunofluorescence
HESCs were seeded in 24-well plates and fixed with 4% PFA. The cells were then permeabilized with 0.3% Triton X-100 in PBS. After washing with PBS, cells were blocked with 5% BSA for 1 h and then incubated with anti-Ki67 antibody (1:1000, Cell Signaling Technology) overnight at 4˚C. Non-specific IgG was used as negative control. After washing with PBS three times, cells were incubated with Anti-mouse IgG (Alexa Fluor® 594 Conjugate) (Cell Signaling Technology) at room temperature for 1 h. Then, 4,6-diamino-2-phenylindole (1 μg/mL, Abcam) was used to stain the nuclei.
Cell counting kit-8 assay (CCK8) and EdU (5-ethynyl-2'-deoxyuridine) assay
The HESCs were seeded in 96-well plates at a density of 3000 cells/well after transfection with siRNAs or lentivirus for 24 h. Optical density (OD) values at 450 nm were measured at 0, 24, 48, 72, and 96 h using a Cell Counting Kit (40203ES80, YEASEN) according to the manufacturer’s instructions.
HESCs were incubated with 50 μM 5-ethynyl-2’-deoxyuridine (Cell-Light EdU Apollo488 In Vitro Kit, RIBOBIO, Guangzhou, China) for 2 h at 37˚C and then fixed with 4% PFA. Cells were stained with fluorescent dyes according to the manufacturer’s instructions. A Leica DMi8 was used to capture images and Image J (NIH) was used to analyze the proportion of positively stained cells. To be specific, the image to be counted was opened in the Image J and converted to greyscale before proceeding. Image-Adjust-Threshold buttons were clicked to highlight the structures and then ‘Apply’ it. If the particles have merged together, Process-Binary-Watershed buttons were clicked to accurately cut them apart. Finally, ‘Analyze Particles’ button was clicked to count cells and data were imported into Excel for calculation.
Flow cytometry analysis for cell cycle assay
After transfection with siRNA or lentivirus for 48 h, the cells were digested with trypsin and washed twice with PBS. Then cells were resuspended in 500 μL of 1× staining buffer containing 10 μL propidium iodide (YEASEN, China). After incubation in the dark at 37˚C for 30 min, flow cytometry analysis was carried out using BD FACSCelesta. Initial cell population gating is placed on FSC-A(rea) vs SSC-A(rea) (cell size vs granularity) (Gate R1). Gate R1 is to remove cell debris. This cell population gate is then placed on FSC-A(rea) vs FSC-H(eight) plot (Gate R2). Gate R2 is then placed on FSC-H(eight) vs FSC-W(idth) plot (Gate R3). Gate R2 and R3 are used to remove the aggregates. Gate R3 is then displayed as a histogram using PI parameter.
The cell cycle status was evaluated using ModFit software.
Wound-healing and transwell assay
For the wound-healing assay, the cells were seeded in a six-well plate and transfected with siRNA or lentivirus for 48 h. When the cells filled the bottom of the plate, a physical gap was created by scraping the monolayers with a pipet tip. Then the cells were washed three times with PBS to remove cell debris and then cultured in DMEM containing 1% CS-FBS. The images were taken with a phase-contrast microscope (Leica) at 0 and 12 h. The area filled with migrated cells was quantified by the Image J software (NIH) through comparing images taken at 0 and 12 h. To be specific, the image was imported into Image J and converted to Type-8 bit. ‘Process-Enhance contrast’ buttons were clicked to make the boundary between the cell edge and the background at the scratch more obvious. Then ‘Process-Find Edge’ buttons were clicked to further greatly enhance the contrast between cells and background. ‘Image-Adjust-Threshold-Apply’ buttons were used to make the background more uniform after binarization. Then magic wand tool was clicked to select the actual scratch area. Finally, ‘Analyze-Measure’ buttons were used to measure the scratch area.
For the transwell assay, cell culture inserts were placed into a 24-well plate. Forty-eight hours after the transfection, 6 × 104 cells/200 µL of DMEM were implanted in the upper chamber of each insert. The lower chambers were filled with 600 µL of DMEM containing 20% CS-FBS, and the cells were incubated at 37˚C for 24 h in normoxic or hypoxic environments. The inserts were washed with ice-cold PBS, and the non-migrating cells were removed from the upper surface of the inserts by wiping with a cotton bud. The cells on the lower surface of the filter were then fixed and stained with 4% PFA and crystal violet. Finally, pictures were taken using an inverted phase-contrast microscope (Leica) after the inserts dried. The number of migrated cells was counted at a magnification of 100×.
RNA-sequencing and downstream bioinformatics analysis
The HESCs were transfected with negative control or siRNA for 24 h. Total RNA from the two groups was reserved using 1 mL of TRIzol (Ambion) and then transferred to Novogene (Beijing, China). An Agilent 2100 bioanalyzer was used for the RNA quality measurements. Sequencing libraries were generated using a NEBNext® UltraTM RNA Library Prep Kit for Illumina®. After the construction of the library, the library was initially quantified by Qubit2.0 Fluorometer and qRT-PCR was used to accurately quantify the effective concentration of the library to ensure the quality of the library. After the library is qualified, the different libraries are then sequenced by the Illumina NovaSeq 6000. FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. DESeq2 R package (1.20.0) was used for differential expression analysis of two groups. The clusterProfiler R package (3.8.1) was used for gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes. The threshold value for significance was P < 0.05, with a fold change >2.
Statistical analysis
A Student’s t-test was used to compare the parametric data between the two groups, and the Mann–Whitney test was used to compare nonparametric data between the two groups. Correlation analysis was performed using the Spearman’s rank correlation test. Statistical significance was set at P < 0.05. At least three independent biological replicates were performed, and data were presented as mean ± s.d. GraphPad Prism 8 was used for graphing and statistical analysis.
Results
The level of TCF3 expression was decreased in the decidua tissues of RPL
TCF3 has been reported to regulate the pluripotency and stemness of embryonic stem cells and plays an important role in embryonic development (Zhang et al. 2013, Brosens et al. 2014). However, its role in RPL remains to be explored. To investigate the role of TCF3 in the pathogenesis of RPL, decidual tissues of RPL patients and HC were collected to detect TCF3 expression using immunohistochemistry (IHC) assay. The results showed that the number of TCF3-positively stained cells was significantly lower in the decidual tissues of RPL patients than in the HC group, and TCF3-positive cells were detected mainly in the stroma (Fig. 1A and B). Further, qRT-PCR analysis showed that the expression of TCF3 mRNA was significantly downregulated in the decidual tissue from RPL patients compared to HC (Fig. 1C). Overall, these results show that TCF3 expression is downregulated in the decidua of RPL patients.
TCF3 expression was downregulated in the decidual tissue in RPL. (A) Representative images of IHC staining for TCF3 in the HC group (n = 8) and in the RPL group (n = 7). The NC was nonspecific mouse IgG. Magnification 200× (Top); scale bar = 50 μm. The boxed areas in the upper images are enlarged and shown in the lower images. (B) Statistical analysis of the percentage of TCF3 positively stained cells in the RPL and HC groups measured by Image J. (C) The mRNA expression level of TCF3 in the decidual tissue from RPL patients (n = 16) and HC (n = 16). *P < 0.05, **P < 0.01. GE: glandular epithelium; HC, healthy control; IHC, immunohistochemistry; NC, negative control; RPL, recurrent pregnancy loss; S, stroma; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
TCF3 expression was downregulated in the decidual tissue in RPL. (A) Representative images of IHC staining for TCF3 in the HC group (n = 8) and in the RPL group (n = 7). The NC was nonspecific mouse IgG. Magnification 200× (Top); scale bar = 50 μm. The boxed areas in the upper images are enlarged and shown in the lower images. (B) Statistical analysis of the percentage of TCF3 positively stained cells in the RPL and HC groups measured by Image J. (C) The mRNA expression level of TCF3 in the decidual tissue from RPL patients (n = 16) and HC (n = 16). *P < 0.05, **P < 0.01. GE: glandular epithelium; HC, healthy control; IHC, immunohistochemistry; NC, negative control; RPL, recurrent pregnancy loss; S, stroma; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
TCF3 expression was downregulated in the decidual tissue in RPL. (A) Representative images of IHC staining for TCF3 in the HC group (n = 8) and in the RPL group (n = 7). The NC was nonspecific mouse IgG. Magnification 200× (Top); scale bar = 50 μm. The boxed areas in the upper images are enlarged and shown in the lower images. (B) Statistical analysis of the percentage of TCF3 positively stained cells in the RPL and HC groups measured by Image J. (C) The mRNA expression level of TCF3 in the decidual tissue from RPL patients (n = 16) and HC (n = 16). *P < 0.05, **P < 0.01. GE: glandular epithelium; HC, healthy control; IHC, immunohistochemistry; NC, negative control; RPL, recurrent pregnancy loss; S, stroma; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
TCF3 knockdown inhibited proliferation and promoted migration in HESCs
To further explore whether TCF3 is involved in the pathogenesis of RPL, we used siRNA to knockdown TCF3 in human endometrial stromal cells (HESCs). The knockdown efficiency was confirmed by qRT-PCR and western blotting (Fig. 2A, B, and C). TCF3 knockdown resulted in a significant decrease in the proliferation of HESCs, as indicated by the Cell Counting Kit-8 (CCK-8) analysis (Fig. 2D). The EdU assay further confirmed that TCF3 knockdown decreased the proportion of EdU-positive cells, which indicated that TCF3 knockdown inhibited HESCs proliferation (Fig. 2E and F). Moreover, immunofluorescence staining for the proliferation marker Ki67 showed similar results (Supplementary Fig. 1A). To further clarify how TCF3 knockdown inhibited HESCs proliferation, flow cytometry assay was performed. The results showed that downregulation of TCF3 led to an increase in the proportion of cells in the G0/G1 phase and a decrease in the percentage of cells in the S phase (Fig. 2G and H). Levels of Cyclin D1, CDK4, and CDK6, which are important cell cycle-related proteins that mediate the transition from G1 to S phase (Topacio et al. 2019), were determined using western blotting in HESCs treated with TCF3 siRNA. The results showed that the expression levels of cyclin D1, CDK4, and CDK6 were decreased in TCF3-knockdown cells (Fig. 2I and J). Together, these results suggest that TCF3 is a key regulator of HESCs proliferation. Previous studies have shown that TCF3 plays an important role in cell migration (Zhao et al. 2013, Miao et al. 2014). To explore the role of TCF3 in HESCs migration, transwell and wound-healing assays were conducted. The wound-healing assay showed that downregulation of TCF3 accelerated wound closure (Fig. 2K and L). In the transwell assay, TCF3 knockdown increased the percentage of migrated cells (Fig. 2M and N). These results suggest that TCF3 knockdown promotes HESCs migration.
Knockdown of TCF3 inhibits proliferation, induces G0/G1 phase arrest and promotes migration in HESCs. (A, B, and C) Verification of the knockdown efficiency of TCF3 in HESCs treated with siRNA against TCF3. (D) CCK-8 assay showing HESCs proliferation at the indicated time. (E and F) EdU assay showing decreased EdU-positive cells in TCF3 knockdown cells. (G and H) Cell cycle assay assessed by flow cytometry analysis. (I and J) The expression of cell cycle-related proteins after TCF3 knockdown. (K, L, M, and N) Effects of TCF3 downregulation on HESC migratory ability. Original magnification, 100×, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Each in vitro test is performed three times, and data are presented as mean ± s.d. HESCs, human endometrial stromal cells; ns, not significant; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
Knockdown of TCF3 inhibits proliferation, induces G0/G1 phase arrest and promotes migration in HESCs. (A, B, and C) Verification of the knockdown efficiency of TCF3 in HESCs treated with siRNA against TCF3. (D) CCK-8 assay showing HESCs proliferation at the indicated time. (E and F) EdU assay showing decreased EdU-positive cells in TCF3 knockdown cells. (G and H) Cell cycle assay assessed by flow cytometry analysis. (I and J) The expression of cell cycle-related proteins after TCF3 knockdown. (K, L, M, and N) Effects of TCF3 downregulation on HESC migratory ability. Original magnification, 100×, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Each in vitro test is performed three times, and data are presented as mean ± s.d. HESCs, human endometrial stromal cells; ns, not significant; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
Knockdown of TCF3 inhibits proliferation, induces G0/G1 phase arrest and promotes migration in HESCs. (A, B, and C) Verification of the knockdown efficiency of TCF3 in HESCs treated with siRNA against TCF3. (D) CCK-8 assay showing HESCs proliferation at the indicated time. (E and F) EdU assay showing decreased EdU-positive cells in TCF3 knockdown cells. (G and H) Cell cycle assay assessed by flow cytometry analysis. (I and J) The expression of cell cycle-related proteins after TCF3 knockdown. (K, L, M, and N) Effects of TCF3 downregulation on HESC migratory ability. Original magnification, 100×, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Each in vitro test is performed three times, and data are presented as mean ± s.d. HESCs, human endometrial stromal cells; ns, not significant; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
Overexpression of TCF3 promoted proliferation and inhibited migration in HESCs
To further study the role of TCF3 in the regulation of HESCs proliferation and migration, TCF3-overexpression lentivirus was transduced to HESCs. The overexpression efficiency was determined by qRT-PCR and western blotting (Fig. 3A, B, and C). The CCK8 assay showed that TCF3 overexpression promoted cell proliferation (Fig. 3D). The EdU experiment obtained a similar result (Fig. 3E and F). The Ki67 assay showed that the percentage of Ki67-positive cells was increased in TCF3-overexpressed cells (Supplementary Fig. 1B). Further, the cell cycle assay demonstrated that TCF3 overexpression promoted the transition from the G0/G1 phase to the S phase (Fig. 3G and H). Moreover, TCF3 overexpression increased the protein levels of cyclin D1, CDK4, and CDK6 (Fig. 3I and J). Wound-healing and transwell assays were also performed to evaluate the migratory ability of HESCs. In TCF3-overexpressed cells, wound closure was delayed, and the number of migrated cells decreased (Fig. 3K, L, M, and N). These results show that overexpression of TCF3 promotes cell proliferation and inhibits HESCs migration.
TCF3 upregulation promotes proliferation, G0/G1–S phase transition and inhibits migration in HESCs. (A, B, and C) The mRNA and protein levels of TCF3 after TCF3 lentiviral transduction. (D) CCK-8 assay showing HESCs proliferation ability at five different time points. (E and F) EdU assay indicating HESCs proliferation ability after TCF3 overexpression. (G and H) The proportion of HESCs in different cell cycle phases is assessed by flow cytometry analysis in TCF3-overexpressing HESCs. (I and J) The expression of cell cycle-related proteins after TCF3 upregulation. (K, L, M, and N) Wound-healing and transwell assays in TCF3-overexpressing HESCs. *P < 0.05; **P < 0.01; ***P < 0.001. HESCs, human endometrial stromal cells; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
TCF3 upregulation promotes proliferation, G0/G1–S phase transition and inhibits migration in HESCs. (A, B, and C) The mRNA and protein levels of TCF3 after TCF3 lentiviral transduction. (D) CCK-8 assay showing HESCs proliferation ability at five different time points. (E and F) EdU assay indicating HESCs proliferation ability after TCF3 overexpression. (G and H) The proportion of HESCs in different cell cycle phases is assessed by flow cytometry analysis in TCF3-overexpressing HESCs. (I and J) The expression of cell cycle-related proteins after TCF3 upregulation. (K, L, M, and N) Wound-healing and transwell assays in TCF3-overexpressing HESCs. *P < 0.05; **P < 0.01; ***P < 0.001. HESCs, human endometrial stromal cells; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
TCF3 upregulation promotes proliferation, G0/G1–S phase transition and inhibits migration in HESCs. (A, B, and C) The mRNA and protein levels of TCF3 after TCF3 lentiviral transduction. (D) CCK-8 assay showing HESCs proliferation ability at five different time points. (E and F) EdU assay indicating HESCs proliferation ability after TCF3 overexpression. (G and H) The proportion of HESCs in different cell cycle phases is assessed by flow cytometry analysis in TCF3-overexpressing HESCs. (I and J) The expression of cell cycle-related proteins after TCF3 upregulation. (K, L, M, and N) Wound-healing and transwell assays in TCF3-overexpressing HESCs. *P < 0.05; **P < 0.01; ***P < 0.001. HESCs, human endometrial stromal cells; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
Downregulation of TCF3 activated the MAPK signaling pathway
The above results have shown the regulatory effect of TCF3 on HESCs. To further explore the mechanism through which TCF3 regulates HESCs proliferation and migration, an RNA-sequencing assay was performed in HESCs treated with TCF3 siRNA or control siRNA. The results found a total of 496 differentially expressed genes. Among them, 305 genes were downregulated and 191 genes were upregulated (P < 0.05 and |log2Foldchange| > 2) (Fig. 4A and B). Further, enrichment analysis was performed using genes with adjusted P-values less than 0.05. The KEGG results showed significantly enriched terms, including cancer pathways, the mitogen-activated protein kinase (MAPK) signaling pathway, and cytokine–cytokine receptor interaction (Fig. 4C). Previous studies have reported that the MAPK pathway plays an important role in cell migration and proliferation (Rousseau et al. 1997, Zhang & Liu 2002). Gene Set Enrichment Analysis further clarified that the MAPK-targeted gene term was enriched when TCF3 was silenced (Fig. 4D). Therefore, we focused on the two main components of the MAPK pathway, p38 and ERK. Western blotting results showed no significant differences in the expression of ERK after TCF3 knockdown or overexpression of TCF3 (Supplementary Fig. 2A and B). However, the levels of phosphorylated p38 (p-p38) protein were increased after TCF3 knockdown, while overexpression of TCF3 decreased p-p38 (Fig. 4E, F, G, and H). These results suggest that TCF3 may regulate the function of HESCs via p38 pathway.
TCF3 downregulation affects the p38 signaling pathway. (A and B) Heatmap and volcano plots indicate differentially expressed genes after TCF3 downregulation. (C and D) KEGG and GSEA (NES = −1.25, P < 0.001) enrichment analysis. (E, F, G, and H) Western blot measurement of p-p38 and p38 after TCF3 downregulation or overexpression. *P < 0.05; **P < 0.01; GSEA, Gene Set Enrichment Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; ns, not significant; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
TCF3 downregulation affects the p38 signaling pathway. (A and B) Heatmap and volcano plots indicate differentially expressed genes after TCF3 downregulation. (C and D) KEGG and GSEA (NES = −1.25, P < 0.001) enrichment analysis. (E, F, G, and H) Western blot measurement of p-p38 and p38 after TCF3 downregulation or overexpression. *P < 0.05; **P < 0.01; GSEA, Gene Set Enrichment Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; ns, not significant; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
TCF3 downregulation affects the p38 signaling pathway. (A and B) Heatmap and volcano plots indicate differentially expressed genes after TCF3 downregulation. (C and D) KEGG and GSEA (NES = −1.25, P < 0.001) enrichment analysis. (E, F, G, and H) Western blot measurement of p-p38 and p38 after TCF3 downregulation or overexpression. *P < 0.05; **P < 0.01; GSEA, Gene Set Enrichment Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; ns, not significant; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
Hypoxia regulated HESCs migration and proliferation via inhibiting TCF3 expression
A previous study has been found that a severe hypoxic environment may play a role in RPL by inhibiting angiogenesis (Fang et al. 2013). To investigate whether hypoxia affects RPL through regulating TCF3 expression, we cultured HESCs in a hypoxic incubator (1% O2) and a normoxic incubator (21% O2) for 48 h. The level of TCF3 protein decreased under hypoxic conditions (Fig. 5A and B). A previous study has found that HIF1A is a central regulator of hypoxic environments (Chang et al. 2018). Therefore, we used siRNA to knockdown HIF1A expression. qRT-PCR and western blotting results showed that TCF3 expression was upregulated when HIF1A was knocked down (Fig. 5C, D, E, and F). Furthermore, the rescue experiment showed when HIF1A siRNA was transfected into HESCs after hypoxic induction, TCF3 expression increased again (Fig. 5G). To further investigate whether hypoxia regulates proliferation and migration in HESCs via TCF3, we used the EdU and Transwell assays to analyze the effect of TCF3 overexpression on HESCs proliferation and migration after hypoxic induction for 48 h. These results showed that hypoxia promoted HESCs migration and inhibited HESCs proliferation. Interestingly, HESCs proliferation was successfully recovered, and migration was inversely inhibited after TCF3 overexpression (Fig. 5H, I, J, and K). Together, these data indicate that hypoxia regulates HESCs migration and proliferation via the HIF1A/TCF3 signaling pathway.
Hypoxia regulates HESCs migration and proliferation through HIF1A/TCF3 signaling axis. (A and B) Western blot measurement of HIF1A and TCF3 expression levels after hypoxic induction for 48 h. (C, D, E, and F) The mRNA and protein levels of TCF3 in HESCs treated with siRNA against HIF1A. (G) The protein level of HIF1A and TCF3 in HESCs transfected with siRNA against HIF1A under hypoxic induction for 48 h. (H and I) The transwell assay shows that TCF3 overexpression reversed the increased migratory ability in HESCs led by hypoxia. (J and K) The EdU assay indicated that overexpression of TCF3 recovered the inhibited proliferation ability of HESCs caused by hypoxia. HESCs, human endometrial stromal cells; HIF1A, hypoxia-inducible factor-1α; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
Hypoxia regulates HESCs migration and proliferation through HIF1A/TCF3 signaling axis. (A and B) Western blot measurement of HIF1A and TCF3 expression levels after hypoxic induction for 48 h. (C, D, E, and F) The mRNA and protein levels of TCF3 in HESCs treated with siRNA against HIF1A. (G) The protein level of HIF1A and TCF3 in HESCs transfected with siRNA against HIF1A under hypoxic induction for 48 h. (H and I) The transwell assay shows that TCF3 overexpression reversed the increased migratory ability in HESCs led by hypoxia. (J and K) The EdU assay indicated that overexpression of TCF3 recovered the inhibited proliferation ability of HESCs caused by hypoxia. HESCs, human endometrial stromal cells; HIF1A, hypoxia-inducible factor-1α; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
Hypoxia regulates HESCs migration and proliferation through HIF1A/TCF3 signaling axis. (A and B) Western blot measurement of HIF1A and TCF3 expression levels after hypoxic induction for 48 h. (C, D, E, and F) The mRNA and protein levels of TCF3 in HESCs treated with siRNA against HIF1A. (G) The protein level of HIF1A and TCF3 in HESCs transfected with siRNA against HIF1A under hypoxic induction for 48 h. (H and I) The transwell assay shows that TCF3 overexpression reversed the increased migratory ability in HESCs led by hypoxia. (J and K) The EdU assay indicated that overexpression of TCF3 recovered the inhibited proliferation ability of HESCs caused by hypoxia. HESCs, human endometrial stromal cells; HIF1A, hypoxia-inducible factor-1α; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
HIF1A was increased in decidual tissue from RPL patients
We investigated the expression level of HIF1A in decidual tissue from RPL patients and HC. The results of qRT-PCR showed that HIF1A mRNA expression was increased significantly in decidual tissue from RPL patients compared with those of HC (Fig. 6A). Western blotting results showed that RPL patients had a higher HIF1A protein level (Fig. 6B). Further, IHC staining was performed to investigate the localization and expression levels of HIF1A. The percentage of HIF1A positively stained cells was significantly upregulated in the stroma and glandular epithelium of RPL patients compared with HC (Fig. 6C and D). Further, linear correlation analysis showed that the HIF1A mRNA level correlated negatively with the TCF3mRNA level in the decidual tissue in RPL and HC (Fig. 6E). Besides, expression of TCF3 was decreased in decidual tissue of RPL compared to that of HCs, while expression of p-p38 was increased in decidual tissue of RPL compared to that of HCs (Fig. 6F).
Increased HIF1A expression in the decidual tissue of RPL patients compared with that of HC. (A) The relative mRNA level of HIF1A in decidual tissue of RPL (n = 16) compared to HC (n = 16). (B and C) Western blotting measurement of HIF1A level in the RPL and HC groups (n = 6). (D and E) Representative images and statistical analysis of IHC staining for HIF1A in the RPL (n = 7) and HC (n = 7) groups. The NC was nonspecific rabbit IgG. Magnification 200× (top), scale bar = 50 μm. The boxed areas in the upper images are enlarged and shown in the lower images. *P < 0.05, ****P < 0.0001 (F) HIF1A mRNA expression level was negatively correlated with the level of TCF3 mRNA expression in decidual tissue in RPL (n = 16) and HC (n = 16). (G) Western blotting measurement of TCF3 and p-p38 level in the RPL and HC groups (n = 6). GE, glandular epithelium; HC, healthy control; HIF1A, hypoxia-inducible factor-1α; NC, negative control; RPL, recurrent pregnancy loss; S, stroma; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
Increased HIF1A expression in the decidual tissue of RPL patients compared with that of HC. (A) The relative mRNA level of HIF1A in decidual tissue of RPL (n = 16) compared to HC (n = 16). (B and C) Western blotting measurement of HIF1A level in the RPL and HC groups (n = 6). (D and E) Representative images and statistical analysis of IHC staining for HIF1A in the RPL (n = 7) and HC (n = 7) groups. The NC was nonspecific rabbit IgG. Magnification 200× (top), scale bar = 50 μm. The boxed areas in the upper images are enlarged and shown in the lower images. *P < 0.05, ****P < 0.0001 (F) HIF1A mRNA expression level was negatively correlated with the level of TCF3 mRNA expression in decidual tissue in RPL (n = 16) and HC (n = 16). (G) Western blotting measurement of TCF3 and p-p38 level in the RPL and HC groups (n = 6). GE, glandular epithelium; HC, healthy control; HIF1A, hypoxia-inducible factor-1α; NC, negative control; RPL, recurrent pregnancy loss; S, stroma; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
Increased HIF1A expression in the decidual tissue of RPL patients compared with that of HC. (A) The relative mRNA level of HIF1A in decidual tissue of RPL (n = 16) compared to HC (n = 16). (B and C) Western blotting measurement of HIF1A level in the RPL and HC groups (n = 6). (D and E) Representative images and statistical analysis of IHC staining for HIF1A in the RPL (n = 7) and HC (n = 7) groups. The NC was nonspecific rabbit IgG. Magnification 200× (top), scale bar = 50 μm. The boxed areas in the upper images are enlarged and shown in the lower images. *P < 0.05, ****P < 0.0001 (F) HIF1A mRNA expression level was negatively correlated with the level of TCF3 mRNA expression in decidual tissue in RPL (n = 16) and HC (n = 16). (G) Western blotting measurement of TCF3 and p-p38 level in the RPL and HC groups (n = 6). GE, glandular epithelium; HC, healthy control; HIF1A, hypoxia-inducible factor-1α; NC, negative control; RPL, recurrent pregnancy loss; S, stroma; TCF3, transcription factor 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0463
Discussion
Normal establishment and maintenance of pregnancy partly depend on proper proliferative and migratory ability of HESCs. In response to estrogen and progesterone, HESCs undergo proliferation and differentiation to be prepared for embryo implantation and placental formation. Impaired HESCs proliferation and migration are associated with many pregnancy complications, such as implantation failure and RPL (Weimar et al. 2013, Aikawa et al. 2020). Previous studies have been found that EZH2 and DNMT3B could epigenetically silence the expression of TCF3, thus promoting the proliferation of endometrial cancer cells (Gui et al. 2021). However, in DU145 and PC3 prostate cancer cells, silencing of TCF3 inhibits proliferation due to G1 arrest (Patel & Chaudhary 2012). In this study, our data indicated that TCF3 knockdown inhibited HESCs proliferation, while overexpression of TCF3 played the opposite function.
Plenty of studies have found that HESCs of RPL disease had an uncontrolled migratory ability in response to embryos with poor developmental potential, which may be associated with the occurrence of RPL (Weimar et al. 2012, 2013). Our results found that knockdown of TCF3 promoted HESCs migration and TCF3 overexpression inhibited HESCs migration. However, the detailed mechanism of how TCF3 regulates HESC migration remains unclear, we performed an RNA-seq in TCF3-knockdown HESCs. We found that TCF3 knockdown was accompanied by upregulated levels of phosphorylated p38 (p-p38), and overexpression of TCF3 decreased levels of p-p38 protein. Therefore, TCF3 might regulate HESC migration through regulating phosphorylation of p38.
Pregnancy complications ranging from RPL to preeclampsia occur when oxygen is dysregulated (Tuuli et al. 2011). It has been reported that hypoxia may inhibit trophoblast invasion through promoting transforming growth factor-beta3 expression, which may play a role in the pathogenesis of RPL (Zhao et al. 2012). Chen et al.(2016) found that the expression level of HIF1A and the number of microvessels in the decidual tissues of patients with RPL during the peri-implantation period were increased compared with those of the normal fertility group, suggesting that a hypoxic environment may be associated with RPL. This finding seems to contradict the previously accepted theory that embryos are planted under physiologically hypoxic conditions, which are essential for early trophoblast cell development and embryogenesis (Jauniaux et al. 2003). Based on this controversy, we collected decidual tissues from RPL patients and HC to detect the expression level of HIF1A, and found that HIF1A expression in the decidua in the first trimester was increased in RPL group compared to those of the HC group. Further, we found that hypoxia increased HIF1A expression and decreased TCF3 expression, while HIF1A knockdown promoted TCF3 expression. This suggests that TCF3 expression is negatively regulated by HIF1A. Hypoxia inhibited HESC proliferation and promoted HESC migration, while overexpression of TCF3 recovered the ability of HESC proliferation inhibited by hypoxia and inhibited HESC migration induced by hypoxia. Hypoxia (increased HIF1A) may be associated with RPL through impaired proliferation and migration function.
The expression of HIF1A is inconsistent in different parts of the whole placenta, which may explain the controversy. A study has shown that HIF1A is highly expressed in the placenta of mice, but the HIF1A level is very low in the decidua (Schäffer et al. 2006). In addition, Jauniaux and colleagues confirmed that before 11 weeks of gestation the partial pressure of oxygen in the placenta was 2.5 times lower than that in the decidua. The PO2 increased independently at both sites during gestation (Jauniaux et al. 2001). In this study, we focused on the decidua in the first trimester, rather than the whole placenta. Several studies have demonstrated a pronounced positive correlation between the expression of HIF1A and the density of microvessels (Madej et al. 2013, Reijnen et al. 2019). Bruno et al. found that increased blood vessel density in decidua parietalis is associated with RPL in the first trimester (Vailhé et al. 1999).
In summary, our study reveals that increased HIF1A inhibits HESCs proliferation and promotes HESCs migration via theTCF3-p38 signaling axis, which was associated with the pathogenesis of RPL disease. Thus, HIF1A and TCF3 may be potential prognostic and therapeutic targets for RPL.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/REP-21-0463.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the National Key Research and Development Program of China (2018YFC1002800), the National Natural Science Foundation of China (82171669 to Yi Lin, 82071647 to Fu-Ju Tian) and the Shanghai Science and Technology (201409001300 to Fu-Ju Tian), the Shanghai Jiao Tong University Trans-med Awards Research (Major Project) (20210201).
Author contribution statement
X-W W, Y L and F J T conceived the study. X-W W wrote the paper. X-W W, X-Q L, Y C Z, and C-M Q performed the experiments and analyzed the data. Y L provided funding. All authors read and approved the final version of the manuscript.
Acknowledgement
The authors would like to thank Prof Svetoslav Chakarov for proofreading the paper.
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