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Ras p21 protein activator 1 regulates trophoblast function and its association with preeclampsia through the Ras/mitogen-activated protein kinase pathway
*Corresponding author: Xueping Shen, The Medical Laboratory Center, Huzhou Maternity & Child Health Care Hospital, Huzhou, China. zyfby0110@163.com
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Received: ,
Accepted: ,
How to cite this article: Ding Z, Lu Y, Deng Q, Hua K, Shen X. Ras p21 protein activator 1 regulates trophoblast function and its association with preeclampsia through the Ras/mitogen-activated protein kinase pathway. CytoJournal. 2025;22:86. doi: 10.25259/Cytojournal_26_2025
Abstract
Objective:
Ras p21 protein activator 1 (RASA1) plays a crucial role in the placenta. However, its effects and mechanisms of RASA1 on trophoblast function in preeclampsia (PE) are unclear. This study aims to investigate the relationship between the regulation of the Ras/mitogen-activated protein kinase (MAPK) pathway by RASA1 and the function of trophoblast cells in PE.
Material and Methods:
Placental tissues were collected from patients with early-onset PE and late-onset PE and their gestational age-matched normal pregnancies. Western blot analysis and quantitative reverse transcription polymerase chain reaction were employed to assess RASA1 levels in placental tissues and trophoblast cells, as well as Ras activation and p38 MAPK phosphorylation levels in the Ras/MAPK pathway. Wound healing, cell counting kit-8, and Transwell migration and invasion assays and flow cytometry experiments were conducted to detect the proliferation, migration, invasion, and apoptosis capabilities of trophoblast cells.
Results:
RASA1 was significantly overexpressed in the placental tissues of patients with PE (P < 0.05). In cells, it downregulated the activation of Ras and phosphorylation of p38 MAPK (P < 0.05) and reduced proliferation and inhibited migration and invasive capabilities (P < 0.05). Moreover, RASA1 increased the rate of apoptosis, promoted the protein expression levels of cleaved caspase3 and Bax, and inhibited the expression of Bcl2 in cells (P < 0.05). The P38/MAPK inhibitor SB203580 reversed the activation of the Ras/MAPK pathway and the effects on proliferation, migration, invasion, and apoptosis of cells induced by si-RASA1 (P < 0.05).
Conclusion:
The activity of the Ras/MAPK pathway could be inhibited by high RASA1 expression, which suppresses cell invasion, migration, and proliferation and boosts apoptosis. The abnormal regulation of the RASA1–Ras/MAPK axis may be a key factor in the development of PE and, therefore, provides new ideas and clinically effective strategies for the diagnosis and treatment of this condition.
Keywords
Preeclampsia
Ras p21 protein activator 1
Ras/mitogen-activated protein kinase pathway
Trophoblast
INTRODUCTION
Preeclampsia (PE), which is characterized by hypertension, edema, and proteinuria, occurs mainly after the 20th week of pregnancy.[1] In severe cases, it can cause damage to the liver, kidneys, heart, and other organs of pregnant women, often resulting in adverse pregnancy outcomes and being a main cause of maternal and perinatal mortality.[2,3] PE is categorized by the gestational age at onset. Early-onset PE (EOPE) is a condition that arises before the 34th week of pregnancy, whereas late-onset PE (LOPE) appears at or after the 34th week of pregnancy.[4] Although the specific mechanism of PE is unclear, it is associated with the inadequate or excessive invasive capability of trophoblast cells, the increased apoptosis of trophoblast cells, the abnormal development of the placenta, endothelial cell (EC) damage, and the onset of PE.[5,6] The delivery of the placenta is the most effective treatment for the management of PE, indicating the important role played by the placenta in the origin and development of PE. The expression of Ras p21 protein activator 1 (RASA1) in the placenta varies at various stages of pregnancy.[7] Its abnormal changes may be related to PE.[8] Therefore, studying RASA1, which regulates trophoblast cell behavior in the placenta, may enhance our knowledge of the occurrence and evolution of PE, providing potentially important new therapeutic insights and improvements in pregnancy outcomes for patients with PE.
The Ras family is a class of small GTPases that are widely involved in differentiation and invasion and serve as an important component of cellular growth and proliferation signaling pathways.[9] Ras is a protein that attaches to the inside of the cell membrane and can bind to either GTP or GDP molecules. It is in an activated state when bound to GTP and in an inactivated state when bound to GDP.[10] Ras GTPase–activating proteins (GAPs) are enzymes that help control the function of Ras proteins. They participate in turning off Ras signaling by increasing the ability of Ras proteins to convert GTP into GDP. The switch from GTP to GDP inactivates Ras, thus dampening the Ras signaling pathway.[11] RASA1 is part of the Ras–GAP family of proteins and was among the earliest discovered members of this group. It is found on chromosome 5q14.3 and has a molecular weight of approximately 120 kilodaltons.[12] The N-terminal region of the RASA1 protein comprises several important domains: PH, SH2, SH3, and a calcium-linked phospholipid-binding site (also known as CaLB or C2). By contrast, the C-terminal end of RASA1 has a GAP domain that is responsible for its enzyme activity, specifically its ability to catalyze the inactivation of Ras proteins.[13] RASA1 connects with the active version of Ras, known as Ras GTP, using its GAP-related catalytic domain. This interaction increases Ras’s natural ability to convert GTP into GDP, inhibiting the Ras signaling pathway.[14]
The Ras/mitogen-activated protein kinase (MAPK) pathway is involved in the growth, migration, and differentiation of trophoblast cells, which are essential for placental formation and function.[15,16] The Ras/MAPK pathway has been found to be inactive in trophoblast cells from patients with recurrent spontaneous abortion (RSA). Further experiments have demonstrated that activating the Ras/MAPK pathway greatly enhances the growth of a chorionic trophoblast cell line.[17] The Ras/MAPK pathway influences how well trophoblast cells can invade surrounding tissues. If the Ras/MAPK pathway is not functioning correctly in these cells, it can result in RSA.[18] In addition, Zhang et al. confirmed that reductions in H3K27me3 in the promoter region of the RASA1 gene can cause the RASA1 gene to become abnormally active. When RASA1 functions abnormally, it suppresses the Ras/MAPK pathway, potentially causing unexplained RSA (URSA).[7] In summary, the Ras signaling pathway is a well-known pathway. It has a vital role in controlling cell growth and programmed cell death. Its activity is tightly controlled by various regulatory proteins like RASA1. Although most studies on the Ras signaling pathway have centered around its role in cancer[19,20] and URSA,[21] the specific molecular mechanisms by which RASA1 regulates the Ras/MAPK pathway to affect trophoblast function in the PE placenta remain unclear.
In this study, we investigated how RASA1 influences the activity of the Ras/MAPK pathway in a trophoblast cell line. We further analyzed how RASA1 affects the apoptosis, migration, proliferation, and invasion of these cells. The abnormal regulation of the RASA1–Ras/MAPK axis may be a key factor in the development of PE and provide new perspectives and clinical strategies for the diagnosis and treatment of this condition.
MATERIAL AND METHODS
Sample acquisition
This research involved four distinct groups of pregnant women and their placentas. The groups were categorized on the basis of the timing of PE onset and corresponding gestational age: 1. Placentas from pregnant women diagnosed with EOPE (n = 30) were collected following cesarean deliveries that occurred between 24 and 33 weeks of gestation. 2. Placentas from healthy pregnant women who underwent cesarean deliveries due to conditions, such as cervical incompetence, fetal distress, placenta previa, or placental abruption, were collected. These placentas, which were designated as normal (n = 30), were matched for gestational age within the range of 24–33 weeks. 3. Placentas from pregnant individuals diagnosed with LOPE (n = 30) were collected following cesarean deliveries occurring between 34 and 41 weeks of gestation. 4. 34–41 normal pregnancies matched for gestational age (n = 30) placentas obtained from healthy pregnant women who gave birth due to fetal distress, placenta previa, abruption of the placenta or maternal request for cesarean delivery. Table 1 shows the clinical data of the study groups. Specifically, it is diagnosed when a pregnant woman’s blood pressure readings exceed 140/90 mmHg on two occasions at least 4 h apart, along with the detection of more than 0.3 grams of protein in her urine over a 24 h period after 20 weeks of gestation.[22] This study included only nonsmoking women carrying a single baby. Women with certain health issues, such as gestational diabetes, kidney disease, cancer, or other infections, or carrying fetuses with genetic abnormalities were excluded from this study. Placental samples were taken from the central part of the placenta within 15 min after birth, avoiding the amniotic membranes. They were washed with sterilized 0.1% diethyl pyrocarbonate (DEPC) solution to remove any blood, and parts of the tissue meant for RNA and protein analyses were rapidly frozen with liquid nitrogen and stored at −80°C. This study was approved by the Ethics Review Committee (Approval No. 2022-R-016) and strictly adheres to the Declaration of Helsinki.[23] All women who participated signed informed consent forms.
| Variable | LOPE group average (n=30) | Normal 1 group average (n=30) | EOPE group average (n=30) | Normal 2 group average (n=30) |
|---|---|---|---|---|
| Maternal age (years) | 29.99 (25–32) | 30.90 (27–32) | 27.40 (24–28) | 28.44 (26–31) |
| Gestational age (weeks) | 37+1 (35+1–39+1) | 37+7 (36+1–40+0) | 31+1 (29+1–32+2) | 32+2 (30+1–33+0 |
| BMI (kg/m2) | 27.90 (25–30) | 27.20 (23–28) | 23.41 (20–26) | 22.60 (20–23) |
| Systolic blood pressure (mmHg) | 165.85 (150–170) | 130.10 (104–132) | 170.18 (157–180) | 130.10 (104–140) |
| Diastolic blood pressure (mmHg) | 98.20 (95–105) | 75.42 (68–76) | 94.02 (91–98) | 65.32 (60–75) |
| Birth weight (g) | 2498.55 (2000–3180) | 3301.20 (3140–3940) | 1320.02 (1069–2112) | 1985.32 (1298–2800) |
PE: Preeclampsia, EOPE: Early-onset preeclampsia, LOPE: Late-onset preeclampsia, BMI: Body mass index
Cell culture and transfection
The trophoblast cell line HTR-8/Svneo was acquired from the ATCC Cell Bank (ZY-H262, Rockville, Maryland, USA). Cells were grown in Dulbecco’s modified Eagle medium (Invitrogen, Mountain View, CA, USA) containing 15% fetal bovine serum (Gibco, Carlsbad, CA, USA) and kept in an incubator at 37°C with 5% carbon dioxide (CO2). Cells were processed and subjected to late-stage experiments at the logarithmic growth phase. They were seeded in a culture dish and subcultured 3 times until they reached a density of 5 × 105, at which point subsequent experiments can be conducted. Non-contamination was verified through mycoplasma detection and short tandem repeat identification. The P38/MAPK inhibitor SB203580 was acquired from Cell Signaling Technology, USA. This compound was dissolved in dimethyl sulfoxide (DMSO) (D2650, Sigma-Aldrich, St. Louis, MO, USA), then used on cells at a concentration of 10 μmoL/L. For the control group, an equivalent amount of DMSO without SB203580 was employed to ensure that any effect observed was due to the inhibitor and not to DMSO.
The RASA1 plasmid and its control plasmid, as well as the RASA1-specific small interfering RNA (siRNA) and its control siRNA, were all synthesized by GenePharma Company (Shanghai, China). The groups transfected with the RASA1 sequence plasmid, control plasmid, RASA1-specific siRNA, and control siRNA were designated as the RASA1, Empty Vector, si-RASA1, and si-NC groups, respectively.
The primers for si-RASA1 were forward 5'-CAUAGAUCACUAUCGAAAATT-3' and reverse 5'-UUUUCGAUAGUGAUCUAUGAT-3'. Cells were grown without antibiotics in six-well plates and transfected at 70– 80% confluence. Transient transfection was conducted using a 20 nM concentration with Lipofectamine™ 2000 reagent (1168019, ThermoFisher, Waltham, MA, USA) in accordance with the manufacturer’s instructions. After 48 h of transfection, cells were harvested for total RNA and protein extraction for Western blot analysis or quantitative reverse transcription polymerase chain reaction (RT-qPCR). Every experiment in the study was conducted 3 times to confirm the accuracy and reliability of the findings.
RT-qPCR
Each sample was collected from the central region of the placenta. The methods used for extracting total RNA from the samples and converting it into complementary DNA (cDNA) through reverse transcription (11119ES60, Yeasen, Shanghai, China) followed the established protocols detailed in an earlier reference.[24] The quality and integrity of the extracted RNA were checked using agarose gel electrophoresis. Primers were synthesized by Zhejiang Shangya Biotechnology and had the following sequences: RASA1 5'-AATACTTCCACCGACATTGAGA-3' (forward); RASA1 5'-TGTAGGCCACTTATGCTGAACA-3' (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 5'-GGACCTGACCTGCCGTCTAG-3' (forward); and GAPDH 5'-TAGCCCAGGATGCCTTGAG-3' (reverse). cDNA samples (50 ng) were prepared through polymerase chain reaction (PCR) in accordance with the following protocol: initial denaturation at 94°C for 30 s, followed by 38 cycles of 94°C for 4 s, and annealing and extension at 60°C for 35 s. PCR was performed using TB Green® Premix Ex Taq™ Kit (RR071A, BIOSCIENCE, Xianggang, China) on a real-time PCR system (Roche, Shanghai, China). Relative gene expression levels were measured by employing the 2−ΔΔCt method.[25]
Western blot analysis
A total of 50 mg of placental tissue was chopped into small pieces using scissors. Transfected cells were rinsed 3 times with phosphate-buffered saline (PBS). Proteins were extracted from placental tissue and transfected cells using a radioimmunoprecipitation lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) containing 1% phenylmethylsulfonyl fluoride and 1% sodium fluoride for 30 min. Protein samples ranging from 30 μg to 50 μg each were loaded into lanes and resolved through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isolated proteins were blotted onto polyvinylidene fluoride membranes (IPVH00011, Biocentury, Beijing, China). The membranes were incubated in 5% skimmed milk for 2 h at room temperature to prevent nonspecific binding. They were then washed 3 times with Tris-buffered saline containing Tween-20, with each wash lasting for 10 min. The membranes were then incubated overnight at 4°C with the following primary antibodies: Anti-RASA1 (1:1000, ab40677), anti-Ras (1:2000, ab52939), anti-P-p38 MAPK (1:2000, ab60999), anti-p38 MAPK (1:2000, ab308333), anti-cleaved caspase3 (1:1000, ab2302), anti-Bcl2 (1:1000, ab182858), anti-Bax (1:1000, ab32503), and anti-glyceraldehyde-3-phosphate dehydrogenase (1:2000, ab8245), all of which were purchased from Abcam (Cambridge, UK). The membranes were treated with secondary antibodies (ab7090, Abcam, Cambridge, UK) that are linked to horseradish peroxidase, specific to the species from which the primary antibodies were derived. These antibodies were used at a dilution of 1:3000 and incubated for 1 h at room temperature. Subsequently, enhanced chemiluminescence (32106, Pierce, Waltham, MA, USA) was utilized to visualize protein bands, which were detected with an imaging system (ChemiDoc XRS, 1708265, Bio-Rad, California, USA). The expression levels of proteins were measured through comparison with those of GAPDH, a standard reference protein, and evaluated using ImageJ (https://imagej.nih.gov/ij/) software for accurate quantification.
Cell counting kit-8 (CCK-8) assay
CCK-8 from Abcam (ab228554, Cambridge, UK) was employed to assess cell growth. Cells were placed in a 96-well plate at a density of 2 × 103 cells per well. Following transfection, cells were incubated for different periods: 0, 24, 48, and 72 h. A total of 15 μL of CCK-8 reagent was introduced to each well at 4 h before the end of each incubation period to measure cell proliferation. The optical density values at 445 nm, which indicate proliferation, were then measured using a microplate reader (MODEL680, Molecular Devices, Sunnyvale, Silicon Valley, USA).
Wound healing assay
After cells were incubated for 24 h under 5% CO2 at 37°C, the bottom of each well was scratched with a sterile 10 μL pipette tip, creating a cell-free wound approximately 1 mm in width. The wells were then rinsed 3 times with PBS to ensure the complete removal of cells. After the addition of fresh serum-free culture medium for further incubation, the migration of cells at the edge of the scratch was examined at designated time points (0 and 48 h) under an inverted microscope (Leica, Germany) and photographed. The relative distance of scratch closure was measured, and scratch closure rate was calculated using the following formula: scratch healing rate (%) = (distance at 0 h−distance at 48 h)/distance at 0 h × 100%.
Transwell migration and invasion assays
After transfecting cells with various constructs (empty vector, RASA1, si-RASA1, and si-NC), 1.5 × 105 cells were resuspended in 250 μL of serum-free Roswell Park Memorial Institute-1640 (RPMI-1640) medium. They were then added to the upper chambers of a Transwell plate with 8 μm pores (Sigma-Aldrich, St. Louis, MO, USA). In the migration assay, the lower chambers of the Transwell plate contained 800 μL of RPMI-1640 with 10% fetal bovine serum to attract cells. In the invasion assay, the upper chambers of the Transwell plate were precoated with 120 μL of diluted Matrigel (diluted 0:8; Sigma-Aldrich, St. Louis, MO, USA) to mimic the extracellular matrix, and the lower chamber contained 750 μL of RPMI-1640 with 20% fetal bovine serum to serve as a chemoattractant. Cells that had invaded or migrated to the underside of the membrane were immobilized with 4% paraformaldehyde for 35 min and stained with 0.1% crystal violet (M07174, Balb, Beijing, China) for 60 min at room temperature. Stained cells were then imaged using a microscope (IX51, Olympus, Tokyo, Japan) and enumerated under 100× magnification.
Flow cytometry
The manufacturer’s instructions for the Annexin V-FITC apoptosis detection kit (product code C1062L, Beyotime, Beijing, China) were followed. Cells were plated in six-well plates, then digested with 0.25% trypsin the next day to prepare cell suspensions. The plates were washed twice with PBS, then applied with 1× binding buffer and 15 μL of FITC-labeled Annexin V. The mixture was incubated for 30 min in the dark. Following centrifugation, cell pellets were resuspended in 1× binding buffer and applied with 10 μL of PI solution. Apoptosis was subsequently detected through flow cytometry (FC-500, Beckman, Fullerton, CA, USA).
Statistical analysis
The Statistical Package for the Social Sciences Statistics 29 software (IBM, NY, USA) was used to analyze the data, with at least three replications for each experiment. Data are shown as the mean ± standard deviation. Comparisons between two groups were performed using the t-test. Comparisons between multiple groups were conducted through one-way analysis of variance, followed by Tukey’s post hoc test. Statistical significance was defined as P < 0.05.
RESULTS
RASA1 was highly expressed in the placental tissues of patients with PE
We analyzed RASA1 gene expression in placental samples from 60 women with PE and 60 gestational age-matched healthy women using RT-qPCR and Western blot analysis to examine how RASA1 is expressed in normal and PE placental tissues. The results revealed that RASA1 messenger RNA (mRNA) levels were markedly higher in the EOPE group than in the normal group (P < 0.05). Similarly, RASA1 mRNA levels were also elevated in LOPE compared to their corresponding control group (P < 0.05) [Figure 1a and b]. The protein levels of RASA1 followed the same pattern, with higher expression in early and LOPE cases than in their corresponding controls (P < 0.05) [Figure 1c and d].
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RASA1 levels in human placental tissues. (a and b) The relative expression levels of RASA1 mRNA were compared between normal and PE placentas. (c and d) The RASA1 protein levels were evaluated using western blot analysis, comparing normal placental tissue to that from PE cases. Each experiment was repeated 3 times. ✶P < 0.05 versus normal 1; ##P < 0.01 versus normal 2. RASA1: Ras p21 protein activator 1, PE: Preeclampsia, mRNA: Messenger RNA, EOPE: Early-onset preeclampsia, LOPE: Late-onset preeclampsia, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
Furthermore, our Pearson correlation analysis demonstrated that RASA1 expression was positively correlated with PE [Table 2]. These findings indicate that the occurrence of PE may be associated with the high expression of RASA1.
| n | RASA1 (Mean±SD) | P-value | Pearson correlation | |
|---|---|---|---|---|
| Normal 1 | 30 | 0.37±0.03 | <0.05 | 0.989 |
| LOPE | 30 | 0.95±0.05 | (<0.000) | |
| Normal 2 | 30 | 0.50±0.04 | <0.01 | 0.993 |
| EOPE | 30 | 1.52±0.08 | (<0.000) |
SD: Standard deviation, LOPE: Late-onset preeclampsia, EOPE: Early-onset preeclampsia
RASA1 inhibited the Ras/MAPK pathway in trophoblast cells
We transfected HTR-8/SVneo cells with RASA1 expression plasmids and specific siRNA to regulate their RASA1 expression. The protein and mRNA expression levels in the RASA1 group were significantly higher than those in the Empty Vector group (P < 0.01) [Figure 2a-c]. Compared with the si-NC group, the si-RASA1 group showed reduced RASA1 expression (P < 0.01) [Figure 2a-c]. Compared with the Empty Vector group, the RASA1 group exhibited marked reductions in the expression levels of active Ras and phospho-p38 MAPK proteins (P < 0.01). By contrast, compared with the si-NC group, the si-RASA1 group presented marked increases in the expression levels of active Ras and P-p38 MAPK proteins (P < 0.05) [Figure 2d-f]. These results reveal that in trophoblast cells, the RASA1 protein inhibits the Ras/MAPK pathway.
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RASA1 inhibited the Ras/MAPK pathway in cells. (a and b) The effects of introducing the RASA1 plasmid and si-RASA1 on the levels of RASA1 protein in cells were evaluated through Western blot analysis. (c) The effect of the RASA1 plasmid and si-RASA1 on the expression of RASA1 mRNA in cells was measured through RT-qPCR. (d-f) The effect of RASA1 on the protein expression levels of active Ras and phospho-p38 MAPK was assessed through Western blot analysis. Each experiment was repeated 3 times. ✶✶P < 0.01 versus RASA1; #P < 0.05, ##P < 0.01 versus si-RASA1. RASA1: Ras p21 protein activator 1, si-RASA1: Small interfering Ras p21 protein activator 1, MAPK: Mitogen-activated protein kinase, mRNA: Messenger RNA, RT-qPCR: Quantitative reverse transcription polymerase chain reaction, si-NC: Small interfering negative control.
RASA1 inhibited the proliferation ability of trophoblast cells and wound healing
We utilized the CCK-8 assay to measure cell proliferation. The assay results revealed that cells transfected with the RASA1 plasmid had a markedly lower proliferation rate than those transfected with the empty vector (P < 0.05) [Figure 3a]. We employed the scratch test to evaluate the wound-healing ability of cells. Its results revealed that after 48 h, the scratch in the RASA1 group was significantly wider than that in the Empty Vector group (P < 0.05) [Figure 3b and c]. Furthermore, in contrast to that in the si-NC group, the cell proliferation rate in the si-RASA1 group significantly increased (P < 0.05) [Figure 3d]. The scratch in the si-RASA1 group was notably narrower than that in the si-NC group (P < 0.05) [Figure 3e and f]. These results suggest that RASA1 reduces the wound-healing capabilities of trophoblast cells.
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RASA1 inhibited the migration and proliferation abilities of cells. (a) The effect of RASA1 on the proliferation of cells. (b and c) The effect of RASA1 on the migration ability of cells. (d) The effect of si-RASA1 on the proliferation of cells. (e and f) The effect of si-RASA1 on the migration ability of cells. (b, e) Scale bar = 100 μm, magnification: 100×. Each experiment was repeated 3 times. ✶P < 0.05 versus RASA1; #P < 0.05 versus siRASA1. RASA1: Ras p21 protein activator 1, si-RASA1: Small interfering Ras p21 protein activator 1, OD: Optical density, NC: Negative control.
RASA1 inhibited the migration and invasion abilities of HTR-8/SVneo cells
We assessed the effect of RASA1 on the invasion and migration abilities of cells using the Transwell assay. Cells with RASA1 exhibited markedly reduced migration and invasion abilities compared with those with the empty vector (P < 0.01) [Figure 4a-c]. Conversely, cells subjected to RASA1 knockdown using si-RASA1 displayed markedly enhanced migration and invasion abilities compared with those in the si-NC group (P < 0.01) [Figure 4d-f]. These findings imply that RASA1 plays a role in inhibiting the movement and invasive behavior of cells.
- Effect of RASA1 on the migration and invasion capabilities of cells. (a-c) The Transwell assay was used to measure migration and invasion capabilities after transfection with either an empty vector and RASA1. (a) Scale bar = 100 μm, magnification: ×100. (d-f) Cells transfected with si-NC and si-RASA1 were evaluated using the Transwell assay. (d) Scale bar = 100 μm, magnification: 100×. Each experiment was repeated 3 times. ✶✶P < 0.01 versus RASA1; ##P < 0.01 versus si-RASA1; ns: No significance. RASA1: Ras p21 protein activator 1, si-RASA1: Small interfering Ras p21 protein activator 1.
RASA1 promoted the apoptosis of HTR-8/SVneo cells
We performed flow cytometry to study apoptosis in cells. Its results revealed that cell apoptosis markedly increased in the RASA1 group compared with that in the Empty Vector group (P < 0.05) [Figure 5a and b]. The expression levels of cleaved caspase3 and Bax were markedly upregulated, but that of Bcl2 was markedly downregulated in the RASA1 group relative to those in the Empty Vector group (P < 0.01) [Figure 5c and d]. In addition, cell apoptosis in the si-RASA1 group significantly decreased compared with that in the si-NC group (P < 0.01) [Figure 5e and f]. The expression levels of cleaved caspase3 and Bax were remarkably down-regulated, and that of Bcl2 was remarkably upregulated in the si-RASA1 group compared with those in the si-NC group (P < 0.05) [Figure 5g and h]. The evidence suggests that RASA1 promotes the apoptosis of cells.
- Effect of RASA1 on the apoptosis of cells. (a and b) The distribution and percentage of cells undergoing apoptosis were compared between the Empty Vector and RASA1 groups. (c and d) The effects of RASA1 on the protein expression levels of cleaved caspase3, Bcl2, and Bax in cells were determined using Western blot analysis. (e and f) The distribution and percentage of apoptotic cells were examined and compared between the si-NC and si-RASA1 groups. (g and h) The effects of si-RASA1 on the protein expression levels of cleaved caspase3, Bcl2, and Bax in cells were determined through Western blot analysis. Each experiment was repeated 3 times. ✶P < 0.05; ✶✶P < 0.01. RASA1: Ras p21 protein activator 1, si-RASA1: Small interfering Ras p21 protein activator 1.
RASA1 regulated the function of trophoblast cells through the Ras/MAPK pathway
The use of the P38/MAPK inhibitor SB203580 reversed the activating effect of si-RASA1 on the Ras/MAPK pathway. The protein expression levels of active Ras and P-p38 MAPK/p38 MAPK in the si-RASA1+SB203580 cotreatment group had significantly decreased compared with those in the siRASA1+NC group (P < 0.05). SB203580 can counteract the activating effect of si-RASA1 on the Ras/MAPK pathway (P < 0.05) [Figure 6a-c].
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RASA1 regulated the function of cells through the Ras/MAPK pathway. (a-c) The P38/MAPK inhibitor SB203580 reversed the activating effect of si-RASA1 on the Ras/MAPK pathway. (d) The CCK-8 assay indicated that the Ras/MAPK pathway mediated the regulatory effect of RASA1 on the proliferation capability of cells. (e and f) The scratch test revealed that the Ras/MAPK pathway mediated the regulatory effect of RASA1 on the migration capability of cells. (e) Scale bar = 100 μm, magnification: ×100. (g-i) The Transwell assay demonstrated that the Ras/MAPK pathway mediated the regulatory effect of RASA1 on the invasion and migration capabilities of cells. (g) Scale bar = 100 μm, magnification: ×100. (j and k) Flow cytometry revealed that the RAS/MAPK pathway mediated the regulatory effect of RASA1 on the apoptosis capability of cells. (l-o) The Ras/MAPK pathway mediated the regulation of the protein expression levels of cleaved caspase3, Bcl2, and Bax in cells by RASA1. Magnification: ×100. Each experiment was repeated 3 times. ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001 versus si-RASA1+NC; #P < 0.05, ##P < 0.01, ###P < 0.001 versus si-RASA1+SB203580. Ras p21 protein activator 1, si-RASA1: Small interfering Ras p21 protein activator 1, MAPK: Mitogen-activated protein kinase, CCK-8: Cell counting kit-8, OD: Optical density.
The CCK-8 experiment demonstrated that cell proliferation speed significantly decreased after treatment with siRASA1+SB203580 relative to after treatment with siRASA1+NC (P < 0.01) [Figure 6d]. Similarly, the scratch test indicated a significantly widened scratch distance at 48 h (P < 0.01) [Figure 6e and f]. In addition, compared with the si-RASA1+NC control treatment, the siRASA1+SB203580 cotreatment significantly inhibited the migration and invasion capabilities of trophoblast cells (P < 0.001) [Figure 6g-i] and promoted their apoptosis (P < 0.001) [Figure 6j and k]. The Western blot results showed that compared with the si-RASA1+NC treatment, the si-RASA1+SB203580 cotreatment markedly upregulated the expression levels of cleaved caspase3 and Bax and downregulated that of Bcl2. SB203580 can counteract the inhibitory effect of si-RASA1 on cell apoptosis (P < 0.05) [Figures 6l-o]. These results imply that RASA1 may influence the occurrence of PE by regulating the Ras/MAPK pathway, which, in turn, affects the behavior of cells.
DISCUSSION
Placental development requires the proliferation of cytotrophoblast cells, which create anchoring villi. These cells then penetrate the uterine lining and move into the maternal spiral arteries, ensuring proper blood flow and nutrient delivery to the developing fetus. The proliferation abilities of trophoblast cells play a key role in the successful development of the placenta.[26] While excessive invasion by tumor cells can cause cancer, insufficient invasion by trophoblast cells can lead to inadequate placental development. This inadequate invasion can contribute to complications, such as PE.[27] As a cytoplasmic protein, RASA1 has been found to play a key role in the migration and invasion of cells in the cancer microenvironment.[28,29] However, its role in the pathogenesis of PE in the placental environment remains unclear. This study aims to first investigate the expression of RASA1 in PE, then to analyze the effect of RASA1 on Ras/MAPK pathway activity in cells, and further explore the effect of RASA1 on the proliferation, migration, and invasion abilities of trophoblast cells.
RASA1, a crucial member of the Ras–GAP family,[28] enhances the activity of Ras GTP, which converts Ras GTP into Ras GDP, thereby suppressing the Ras signaling pathway. Consequently, RASA1 is a critical regulator of the intracellular signaling pathways that control cell proliferation and apoptosis. RASA1 suppresses cell proliferation and encourages apoptosis.[30]
Research has demonstrated that in the absence of RASA1, dysregulated Ras/MAPK signal transduction in ECs results in the impaired folding of collagen IV and its retention in the endoplasmic reticulum, leading to EC death.[30] Zhang et al. found that MEG3 inhibits the expression of RASA1 by mediating the histone methylation of the promoter of the RASA1 gene by EZH2, thereby activating the Ras/MAPK pathway and enhancing the proliferative and invasive capacities of trophoblasts.[31] Despite these findings, studies on how the RASA1-Ras/MAPK regulatory axis functions in trophoblast cells remain limited. In addition, the potential application of this axis in the clinical treatment of PE remains uncertain and requires further investigation. In our study, the protein and mRNA levels of RASA1 in placentas from women with EOPE and LOPE were markedly higher than those in placentas from healthy pregnancies of the same gestational age. Consistent with previous findings, the upregulation of RASA1 expression in the PE placenta reduces cell invasion and promotes the apoptosis of human trophoblast layer cells.[8] After transfection with the RASA1 plasmid, the protein and mRNA levels in the si-RASA1 group were substantially reduced. Subsequently, cellular experiments analyzing the effect of RASA1 on the activity of the Ras/MAPK pathway in trophoblast cells revealed that RASA1 does indeed have a negative regulatory effect on the Ras/MAPK pathway. This finding aligns with the discovery of high RASA1 expression and Ras/MAPK pathway inactivation in PE placental tissue, indicating a close association between Ras/MAPK pathway inactivation and RASA1 in PE placental tissue. These results are consistent with the role of the RASA1-Ras/MAPK regulatory axis in URSA, whereas RASA1-Ras/MAPK axis exhibits opposite function in ECs.[30,31] In placentas affected by PE, the increased expression of RASA1 can stimulate its intrinsic GTPase activity. This action negatively affects RAS pathways, which are typically involved in promoting cell survival and preventing apoptosis. This action effectively deactivates these pathways and inhibits cell proliferation.[32,33]
This study demonstrated that RASA1 can reduce the proliferation of trophoblast cells, inhibit their migration and invasion abilities, and promote apoptosis. These observations are in line with those of previous research suggesting that RASA1 promotes proliferation and improves migration in various cell types, including rectal cancer,[34] liver cancer, and cutaneous squamous cells.[35] Furthermore, research demonstrated that the inhibitor SB203580, which inactivates the p38 MAPK pathway, can decrease the proliferation of breast cancer cell lines and bladder cancer cell lines.[36,37] This study examined how RASA1 affects trophoblast cell function when the Ras/MAPK pathway is inactivated using the inhibitor SB203580. Its findings reveal that when the activity of the Ras/MAPK pathway is inhibited, the biological effects of si-RASA1 on the function of trophoblast cells remarkably weaken. The above findings demonstrate that siRASA1 regulates trophoblast function by activating the Ras/MAPK pathway. Therefore, the activity of the Ras/MAPK pathway could be inhibited by high RASA1 expression, thereby inhibiting invasion, migration, and proliferation but boosting apoptosis in trophoblast cell lines. Our results verify that the inhibition of Ras/MAPK regulation by high RASA1 expression leads to PE, providing new insights and clinical strategies for the diagnosis and treatment of this complication. However, this study has certain limitations, namely, the use of a single trophoblast cell line and potential off-target effects of SB203580. The role of the upstream regulators of RASA1 in the potential mechanisms of the RASA1–Ras/MAPK axis (including the Ras/Raf/MEK/ERK/JNK signaling pathway) in the placentas and cells of animal models of PE in vivo or in the placentas and primary trophoblast cells of patients with PE and the treatment of PE need to be further investigated.
SUMMARY
The activity of the Ras/MAPK pathway could be inhibited by high RASA1 expression, thereby inhibiting invasion, migration, and proliferation, but boosting apoptosis in trophoblast cell lines. In the future, we will investigate in vivo the role of the upstream regulators of RASA1 and specific regulatory mechanisms of the RASA1-Ras/MAPK axis in the placentas and cells of animal models of PE or in the placentas and primary trophoblast cells of patients with PE.
Our research results verify that the abnormal regulation of the RASA1-Ras/MAPK axis in trophoblast cell lines is closely related to the occurrence and development of PE, providing new insights and clinical strategies for the diagnosis and treatment of this complication.
AVAILABILITY OF DATA AND MATERIALS
The data analyzed were available on the request for the corresponding author.
ABBREVIATIONS
cDNA: Complementary DNA
CO2: Carbon dioxide
DEPC: Diethyl pyrocarbonate
DMSO: Dimethyl sulfoxide
EC: Endothelial cell
EOPE: Early-onset PE
GAPs: GTPase-activating proteins
LOPE: Late-onset PE
MAPK: Mitogen-activated protein kinase
mRNA: Messenger RNA
PBS: Phosphate-buffered saline
PCR: Polymerase chain reaction
PE: Preeclampsia
RASA1: Ras p21 protein activator 1
RSA: Recurrent spontaneous abortion
RT-qPCR: Quantitative reverse transcription polymerase
chain reaction
siRNA: Small interfering RNA
URSA: Unexplained RSA
ACKNOWLEDGMENT
Not applicable.
AUTHOR CONTRIBUTIONS
ZYD: Designed the research study; YL and QXD: Performed the research; KH and XPS: Provided help on the experiments and analyzed. All authors contributed to important editorial changes in the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. All authors read and approved of the final manuscript. All authors meet ICMJE authorship requirements.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
This study has been approved by the Ethics Review Committee of Huzhou Maternity & Child Health Care Hospital (Approval No. 2022-R-016) and strictly adheres to the Declaration of Helsinki. Informed consent was obtained from all the patients.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
EDITORIAL/PEER REVIEW
To ensure the integrity and highest quality of CytoJournal publications, the review process of this manuscript was conducted under a double-blind model (authors are blinded for reviewers and vice versa) through an automatic online system.
FUNDING: This study is supported by Huzhou Science and Technology Plan Funding Project for Public Welfare Application Research Project (2022GYB07).
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