Galectin-14 Promotes Trophoblast Migration and Invasion by Upregulating the Expression of MMP-9 and N-Cadherin

Galectin-14 is specifically expressed in placental trophoblasts, and its expression is reduced in trophoblasts retrieved from the cervix of women destined to develop early pregnancy loss. However, the roles of galectin-14 in regulating trophoblasts and in the pathogenesis of pregnancy complication have never been investigated. In the current research, we aimed to investigate the roles of galectin-14 in the regulation of trophoblasts. Tissues of the placenta and villi were collected. Primary trophoblasts and human trophoblast cell line HTR-8/SVneo were used. Western blotting and RT-PCR were used to quantify gene expression. The siRNA-mediated galectin-14 knockdown and lentivirus-mediated overexpression were performed to manipulate the gene expression in trophoblasts. Transwell migration and invasion assays were used to evaluate cell migration and invasion capacity. Gelatin zymography was used to determine the gelatinase activity. Galectin-14 was significantly decreased in the villi of early pregnancy loss and the placenta of preeclampsia. Knockdown of galectin-14 in primary trophoblasts inhibited cell migration and invasion, downregulated the expression of matrix metalloproteinase (MMP)-9 and N-cadherin, the activity of MMP-9, and decreased the phosphorylation of Akt. Meanwhile, the overexpression of galectin-14 in HTR-8/SVneo promoted cell migration and invasion, upregulated the expression of MMP-9 and N-cadherin, the activity of MMP-9, and increased the phosphorylation of Akt. Increased Akt phosphorylation promoted cell migration and invasion and upregulated the expression and activity of MMP-9, while decreased Akt phosphorylation inhibited cell migration and invasion and downregulated the expression and activity of MMP-9. Thus, galectin-14 promotes trophoblast migration and invasion by enhancing the expression of MMP-9 and N-cadherin through Akt phosphorylation. The dysregulation of galectin-14 is involved in the pathogenesis of early pregnancy loss and preeclampsia.


INTRODUCTION
Placenta is a temporary organ connecting the fetus and her mother. The trophoblast is the main cellular component of the placenta. The normally functioning trophoblast is crucial for the establishment and maintenance of pregnancy, development of the placenta, remodeling of the spiral artery, and subsequently fetal growth and maturation (Turco and Moffett, 2019). Dysregulation of trophoblasts is associated with pregnancy complications including early pregnancy loss (EPL) (Huppertz, 2020) and preeclampsia (Huppertz, 2018).
The function of trophoblasts is regulated by a group of genes, especially placenta-specific genes including PLAC1 and syncytin (Rawn and Cross, 2008). These genes regulate trophoblastic migration, invasion, proliferation, apoptosis, and metabolism (Bolze et al., 2017;Wan et al., 2019). Galectins are among these genes.
Galectin-14, a recently identified galectin family member, is specifically expressed in placenta with over 12,000 times expression level compared with other tissues (Than et al., 2009). Using immunocytochemistry, a decrease in galectin-14 was observed in trophoblasts retrieved from the cervix at the gestational age of 5-10 weeks before the onset of early pregnancy loss (Fritz et al., 2015). However, the regulatory roles of galectin-14 in trophoblasts have never been investigated yet.
In the current study, the placental expressions of galectin-14 in early pregnancy loss and severe preeclampsia were determined, the effects of galectin-14 on the migration and invasion of trophoblast were observed, and mechanisms behind were explored.

Subjects
Villi were collected from 28 women who were undergoing artificial abortion and 28 cases of early pregnancy loss. Placenta tissues were collected from 20 women of normal pregnancy and 20 with severe preeclampsia at Cesarean sections.
Early pregnancy loss was diagnosed if the ultrasound examination showed an empty sac with a mean diameter over 25 mm or an embryo with a crown-rump length over 7 mm without heartbeat within the first 12 6/7 weeks of gestation (American College of Obstetricians and Gynecologists' Committee on Practice Bulletins-Gynecology, 2018). Women undergoing artificial abortions with a similar gestational age served as control. The pregnancies complicated by autoimmune disease, genital malformation, history of exposure to poison or X-ray, and other acute or chronic diseases were excluded. Severe preeclampsia was diagnosed according to the criteria recommended by the American College of Obstetricians and Gynecologists (ACOG) (American College of Obstetricians and Gynecologists, 2019). Exclusion criteria were multiple pregnancy, pregnancy complicated by chronic hypertension, diabetes mellitus, and other acute or chronic diseases. Demographic characteristics of the cases were described in Tables 1, 2. This study was approved by the Institutional Ethical Committee of Women's Hospital, School of Medicine, Zhejiang University, and informed consents were obtained.
Primary extravillous trophoblasts (EVTs) were isolated from the villi of artificial abortion. Briefly, tissues were washed with cold phosphate buffered saline (PBS). Amniotic membrane and decidua were removed, and villi were minced. The fragments were resuspended with high-glucose DMEM with 10% FBS. After the cells attached on the bottom, bottles were washed with PBS gently, and fresh medium was added. EVT purity was examined by immunofluorescence staining of HLA-G and CK-7. Cells with purity over 90% and within four passages were used in the experiments.

Gene Knockdown and Overexpression
Cells were transfected with a SMARTpool siRNA or scramble siRNA (GenePharma, Shanghai, China) using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, Waltham, United States) according to the manufacturer's instructions.
For overexpression, cells were transfected with lentivirus carrying overexpression vector or empty vehicle according to the proper multiplicity of infection (MOI). Here, 5-8 mg/ml polybrene (Cell-Land, Hangzhou, China) was added in the culture medium.

Western Blotting
Tissues were homogenized in 200 µl radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail (Selleck, Houston, United States) on ice. Cells were lysed with 50 µl RIPA lysis buffer containing protease inhibitor cocktail on ice.

RT-PCR and RT-qPCR
RNA isolation and reverse transcription were performed as previously described (Yan et al., 2016).
Reverse transcription semiquantitative PCR (RT-PCR) was performed using a PTC 200 thermal cycler, and each 25-µl reaction contained 1 × GoldStarTaq Master Mix, 100 ng cDNA, and 400 nmol/L (nM) of each specific primer. Amplifications were performed as follows: 10 min at 95 • C, 30 amplification cycles (30 s at 95 • C, 30 s at 55 • C, and 1 min at 72 • C) and final extension for 5 min at 72 • C. PCR products were analyzed by 2% ethidium bromide staining agarose gel electrophoresis, quantified by densitometry (Image Lab software, Bio-Rad Laboratories Inc., Hercules, United States) and normalized against GAPDH.
The quantitative PCR (qPCR) was performed in ViiA 7 Real-Time PCR System (Thermo Fisher Scientific, Waltham, United States) by using TB Green Premix Ex Taq (Tli RNase H Plus) Kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions.

Transwell Migration and Invasion Assays
Cells were harvested and seeded in the upper chamber with serum-free medium. For invasion assay, the upper chamber was precoated with Matrigel (BD, Franklin Lakes, United States). And the lower chamber contained 500 µl complete medium. After 8 h for migrating or 24 h for invading, the bottoms of the inserts were fixed and stained in a solution containing 0.1% Gentian violet and 20% methanol. Cells were photographed (50×, 100×) and counted (100×) with an inverted phase contrast microscope (Leica, Wetzlar, Germany).

Statistical Analysis
Kolmogorov-Smirnov test was used to test the normal distribution of the data. Levene test was used to test the homogeneity of variance. Student t-test and Mann-Whitney test were used for data comparison. p < 0.05 was considered significant. SPSS (International Business Machines Co., Armonk, United States) was used for statistical analysis.

Placental Galectin-14 Is Dysregulated in Early Pregnancy Loss and Preeclampsia
Placenta tissues were obtained from women with early pregnancy loss, preeclampsia, and their controls. Demographic characteristics of the subjects were described in Tables 1, 2.
There were no significant differences in maternal age and the gestational age at curettage between early pregnancy loss and its control. There were no significant differences in maternal age and fetal gender between severe preeclampsia and its control. The significant differences in blood pressure and proteinuria were consistent with the diagnosis of preeclampsia. The differences in gestational age at delivery and neonatal birth weight were due to preeclampsia. The difference in body mass index (BMI) was in accordance with the conclusion obtained in previous research that higher BMI is a risk factor for preeclampsia (Bartsch et al., 2016) ( Table 2). Galectin-14 expression was determined by Western blotting. Placental expression of galectin-14 was significantly lower in both early pregnancy loss and preeclampsia as compared with their controls (Figure 1). The dysregulation of placental galectin-14 in early pregnancy loss and preeclampsia implies its possible role in the pathogenesis of pregnancy complications and in the regulation of trophoblasts.

N-Cadherin and Matrix Metalloproteinase 9 Contribute to the Promotion of Trophoblast Migration and Invasion
To investigate the involvement of N-cadherin and MMP-9 upregulation in the promotion of trophoblast migration and invasion, we performed siRNA-mediated knockdown of N-cadherin and MMP-9 in galectin-14-overexpressed HTR-8/SVneo. The N-cadherin and MMP-9 expression reduced by 75 and 60%, respectively ( Figure 5A). The activity of MMP-9 reduced by 70-90% ( Figure 5B). Knockdown of MMP-9 and N-cadherin decreased migration and invasion of HTR-8/SVneo (Figure 5C).

Akt Signaling Is Involved in Galectin-14 Promotion of Matrix Metalloproteinase 9 Expression
Inhibition of Akt phosphorylation at Ser473 was observed in galectin-14-knockdown primary EVTs, while activation was detected in galectin-14-overexpressed HTR-8/SVneo (Figures 3A,B). Akt phosphorylation agonist (SC79) enhanced the cell migration and invasion ability (Figure 6) and increased MMP-9 expression and activity and MMP-9/TIMP-1 ratio (Figure 7). On the contrary, Akt inhibitor (LY294002) inhibited the cell migration and invasion (Figure 6) and decreased MMP-9 expression and activity and MMP-9/TIMP-1 ratio (Figure 7). No significant change was detected in N-cadherin expression. These findings imply that enhancement of Akt phosphorylation is necessary for the promotion of MMP-9 by galectin-14.

DISCUSSION
The dysfunction of trophoblasts is one of the major pathogeneses of early pregnancy loss and preeclampsia. In the current investigation, we found the decreased placental expression pattern of galectin-14 in early pregnancy loss and preeclampsia, which were consistent with previous studies (Than et al., 2014;Fritz et al., 2015;Balogh et al., 2019). These findings point to the importance of galectin-14 in these pregnancy-related diseases. We further demonstrated that galectin-14 promoted the migration and invasion of trophoblasts through enhancing the expression of MMP-9 and N-cadherin.
In the placenta, galectin-14 is expressed in STB and EVT and is necessary for the maintenance of immune tolerance at the fetal-maternal interface (Than et al., 2009(Than et al., , 2014Balogh et al., 2019). However, little is known about the regulatory effects of galectin-14 on trophoblasts. In this study, the effects of galectin-14 knockdown or overexpression on trophoblast migration and invasion indicate the positive impact of galectin-14 on trophoblast migration and invasion. Successful trophoblast migration and invasion play a key role in embryonic implantation and establishment of healthy pregnancy. Thus, the dysregulation of galectin-14 in trophoblasts is involved in the pathogenesis of early pregnancy loss and preeclampsia. In addition, unlike the recombinant galectin-14 used in the previous researches, our results came from the direct knockdown of endogenic galectin-14 in primary EVTs, which are more convincing. And we further established a galectin-14-synthesizing trophoblast cell line for studies on mechanisms.
Epithelial-mesenchymal transition (EMT) is another indispensable process for trophoblasts obtaining motility and invasion capacity. During EMT, trophoblasts lose their endothelial phenotype, loosen intercellular adhesion, and modulate cytoskeleton. The cell-cell junction proteins, such as E-cadherin, are degraded, while mesenchymal cell markers, such as N-cadherin and VE-cadherin, are upregulated (Kokkinos et al., 2010). The temporal and spatial expression of cadherins is the hallmark of trophoblast phenotype transformation, and defects in this process result in shallow trophoblast invasion, which are associated with pregnancy complications, such as early pregnancy loss and preeclampsia (Brown et al., 2005;Multhaup et al., 2018). In mice, galectin-1 improves trophoblast invasion and migration by increasing N-cadherin expression and decreasing E-cadherin expression (You et al., 2018). Herein, we observed that galectin-14 enhanced the expression of N-cadherin; meanwhile, it prompted the invasion and migration of trophoblasts, indicating that, like galectin-1, galectin-14 facilitates migration and invasion by promoting EMT in trophoblasts.
FIGURE 8 | Schematic representation of galectin-14 enhancing trophoblast migration and invasion. The expression of galectin-14 during trophoblast differentiation promotes Akt phosphorylation, leading to increasing matrix metalloproteinase (MMP)-9 expression, secretion, and activity, thus increasing the degradation of extracellular matrix (ECM). Galectin-14 also promotes epithelial-mesenchymal transition (EMT) by upregulating N-cadherin expression. Together, the increased ECM degradation and EMT help trophoblasts obtain the capacity of migration and invasion. study, we found that galectin-14 promoted the phosphorylation of Akt. Increasing the phosphorylation of Akt promoted trophoblast migration and invasion and MMP-9 expression and activity, but not N-cadherin. These data suggest that galectin-14 enhances MMP-9 expression and activity through promoting Akt phosphorylation, while the mechanism through which galectin-14 promotes N-cadherin expression remains to be studied.
An in vivo research will enhance our insight into the roles of galectin-14 in pregnancy, placental development, and pathogeneses of early pregnancy loss and preeclampsia. However, there is no homologous gene of LGALS14 (the gene of galectin-14) in rat or mouse, which hammered the investigation with animal models.
In summary, galectin-14 promotes trophoblast migration and invasion by enhancing the expression of MMP-9 and N-cadherin through Akt phosphorylation (Figure 8). The dysregulation of galectin-14 is involved in the pathogenesis of early pregnancy loss and preeclampsia.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Institutional Ethical Committee of Women's hospital, School of Medicine, Zhejiang University. The patients/participants provided their written informed consent to participate in this study.