The expression of TUSC3 in Preeclampsia and the function in trophoblast cell

LI Meng-yong-wei, KANG Jin-yu, LIN Dan, SUN Fei, JIE Qiu-ling, MA Yan-lin?

1.Hainan Provincial Key Laboratory for human reproductive medicine and Genetic Research, Hainan Provincial Clinical Research Center for Thalassemia, the Key Laboratory of Tropical Translational Medicine of Ministry of Education, Department of Reproductive Medicine, the First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, Hainan 571101, China

2.Haikou Key Laboratory for Preservation of Human Genetic Resource, the First Affiliated Hospital of Hainan Medical University, Haikou, Hainan 571101, China

Keywords:Preeclampsia TUSC3 Trophoblast cells Migration assay Transwell assay

ABSTRACT

1.Introduction

Preeclampsia (PE) is a specific disease of pregnancy, which refers to a systemic syndrome with hypertension and proteinuria as the main clinical manifestations after 20 weeks of pregnancy,and systemic small vessel spasm, increased vascular permeability and decreased perfusion of various systems or organs as the basic pathophysiological changes in pregnant women.It is an important cause of maternal and perinatal morbidity and mortality[1,2].The pathogenesis of PE has not been clarified so far, and there is no early prediction and precise treatment.Termination of pregnancy is still the most effective treatment.Trophoblast dysfunction and uterine spiral artery remodeling disorder are recognized as the pathophysiological basis of PE at home and abroad[3].Therefore, it is of great clinical significance to explore the key factors regulating trophoblast invasion from the perspective of etiology, aiming at the pathogenesis of PE, and early prediction and intervention of PE.

TUSC3 (tumor suppressor candidate gene 3) gene was located in band 2 of chromosome 8 short arm region 2 (8p22) in 1996 and is highly expressed in a variety of epithelial cells and tissues(including prostate, colon, lung, liver, ovary, testis and adipose tissue)[4,5].With the development of research, more and more important functions of TUSC3 have been discovered.TUSC3 affects magnesium ion (Mg2+) transport by interacting with protein phosphatase 1 (PPPC1A) and is involved in the regulation of human learning and memory[6].TUSC3 also plays an important role in the regulation of essential cellular uptake of magnesium ions (Mg2+)during embryonic development.Researchers also found that TUSC3 is a potential tumor suppressor gene, which is closely related to the occurrence and development of a variety of tumors[7].Our previous study found that TUSC3 regulated the proliferation, migration and invasion of cervical cancer cells by inhibiting the AKT signaling pathway[8].In the public database GSE12767, TUSC3 was found to be significantly upregulated in the first trimester villi of PE compared with controls.Therefore, we speculate that TUSC3 may regulate trophoblast function and participate in the occurrence and development of PE.The aim of this study is to further analyze the expression level of TUSC3 in placentas of PE patients and its effect on trophoblast function, and to further explore the correlation between TUSC3 and the pathogenesis of PE, so as to provide some theoretical basis for early prediction and intervention of clinical PE.

2.Materials and Methods

2.1 General materials

DMEM medium, fetal bovine serum, trypsin and double antibody were purchased from Gibco.Lentiviral TUSC3 overexpression plasmid (pLV[shRNA]:T2A:Puro-U) and control plasmid were purchased from Shanghai Jikai Gene Chemical Technology Co.,LTD.The reverse transcription kit was purchased from Takara, the Transwell cell chamber was from Bio Jet, and the wound healing 2-well insert culture dish was from Ibidi.Trizol lysate was purchased from Invitrogen, and xylene, paraformaldehyde, methanol, and absolute ethanol were purchased from Xi long Scientific.

2.2 Placental tissue samples

Placental tissues were collected from cesarean section in the First Affiliated Hospital of Hainan Medical University from January 2022 to December 2022, including placental tissues from 10 normal pregnant women in the control group and 10 PE patients.Patients were selected strictly according to the inclusion criteria and exclusion criteria.The inclusion criteria were: Women who met the“Obstetrics and Gynecology (9th Ed)” diagnostic criteria for PE had a systolic blood pressure of 140 mmHg or more and/or a diastolic blood pressure of 90 mmHg or more after 20 weeks of gestation,with proteinuria of 300 mg or more per 24 h or random positive proteinuria or without proteinuria but with one or more of the following: Abnormal liver function (serum aminotransferase levels more than 2 times the normal value); Platelet count less than 100×109/L; Pulmonary edema; Abnormal renal function (serum creatinine level ＞ 1.1mg/dl or ＞ 2 times the normal value); New central nervous system abnormalities or visual disturbances.All the pregnant women had no other medical or surgical diseases, and there was no history of heavy medication, smoking and drinking during pregnancy.At the same time, the clinical indicators of the two groups were collected,such as maternal age, reproductive history, gestational age at onset of PE, gestational age at delivery, systolic blood pressure, diastolic blood pressure, urine protein, neonatal gender, neonatal weight,placental weight, etc.The specimen collection had been approved by the Ethics Committee of the First Affiliated Hospital of Hainan Medical College, and all enrolled patients had signed an informed consent.

2.3 Cell lines and cell lines overexpressing TUSC3

Human chorionic trophoblast cell line HTR8/SVneo (hereinafter referred to as HTR8) was purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences.It is a normal trophoblast cell line with EVT phenotype.Cell culture conditions were DMEM(C11995500BT, Gibco, USA)+10%FBS(Gibco)+1% penicillin/streptomycin at 37 ℃, 5%CO2.Retrovirus was coated into 293T cells by calcium phosphate method.Forty-eight hours after transfection, the medium supernatant containing virus was collected and centrifuged at 300 g for 3 min to remove the supernatant.5×virus concentrate was added, precipitated overnight in a refrigerator at 4 ℃, and centrifuged at 300 g for 15 min at centrifugal force.The supernatant was removed, and the virus was resuspended in PBS to precipitate the transfected cells to establish a cell line with stable overexpression of TUSC3.

2.4 Total RNA extraction and qRT-PCR

Total RNA was extracted from placental tissue using Trizol method(Invitrogen), and the purity and concentration of RNA were detected.Then cDNA was synthesized and quantified by PrimeScriptTM-RT reagent Kit (Takara).The reaction solution was prepared according to the instructions of SYBR Premix Ex Taq TMI Ⅱ of Takara Company.The amplification conditions were 95 ℃ for 30s, 95℃ for 5s, 55 ℃ for 30s, 72 ℃ for 30s, 45 cycles.The dissolution curve and cycle threshold (CT value) were analyzed.The 2-ΔΔCtmethod was used for calculation and statistical analysis.The primer sequences are shown in Table 1.

2.5 Immunohistochemistry

Some normal and PE placentas were fixed with 4%paraformaldehyde and routinely prepared tissue sections with athickness of 4-5 μm for immunohistochemical staining (SP).The primary antibody was polyclonal rabbit anti-human TUSC3 antibody(1:500;no.ab230520; Abcam), the secondary antibody was goat anti-rabbit IgG, and the primary antibody was replaced by PBS as negative control.The slides were counterstained with hematoxylin and then sealed with glycerin gelatin before the results were observed under a microscope.

Tab1 Major Primer sequences

2.6 Wound-healing assay

1 mL of cell resuspension was prepared in a volume of 6×105/mL.The dish for scratch experiment was removed, and 70 μL of the above cell suspension was added to the left and right chambers of the central groove of the dish and left for 2-5 min.The small dishes were transferred to an incubator at 37 ℃, and after 12 h of culture,the cell adherence was observed.After cell attachment, the groove was removed with forceps and gently washed twice with 1mL PBS,followed by the addition of 1 mL complete medium.Microscopic photographs were recorded at 0 h at this time and were taken at 6-h intervals thereafter until migration confluence.

2.7 Transwell migration assays

100 μL of diluted Matrigel gel was added to the upper chamber of the Transwell chamber and incubated for 1 h in an incubator at 37℃.Cells were counted and the density was adjusted to 2.5×105/mL.The Matrigel gel in the upper chamber was discarded, and 200 μL of cell suspension was added to the upper chamber of each well, and 600 μL of complete medium was added to the lower chamber, and the cells were cultured in an incubator at 37 ℃ for 24 h.After 24 h of incubation, the upper chamber medium was discarded, the upper chamber cells were gently wiped off with a cotton swab, washed with PBS, and repeated twice.The medium of the lower chamber was aspirated and discarded and washed three times with PBS.700

μL of 4% paraformaldehyde was added to each well and fixed for 30 min at room temperature in equilibrium.The fixative solution was removed and washed 2-3 times with PBS to prepare Giemsa staining solution (1 mL Giemsa stock solution +7 mL ddH2O).700 μLGiemsa staining solution was added to each well and stained for 10-30 min.The staining time was adjusted according to the cell staining observed under the microscope.Staining was terminated by rinsing with running water and allowed to dry before being photographed and counted under a microscope.

2.8 Statistical Analysis

Statistical software SPSS23.0 was used for all statistical analyses.In clinical indicator analysis,EM was generally used for data analysis.Normality test was performed before data analysis.T-test was used to analyze the comparison between groups of measurement data with normal distribution, rank sum test was used to analyze the comparison between groups of measurement data with non-normal distribution, and Chi-square test was used to analyze the comparison between groups of counting data, P＜0.05 was considered to indicate statistical significance.

3.Results

3.1 Analysis of clinical indicators of patients

We collected the clinicopathological information of 10 cases of PE group and normal control group for statistical analysis.Compared with the control group, the PE group had significantly higher Systolic blood pressure (SBP) and Diastolic blood pressure (DBP),and a higher proportion of proteinuria (P＜0.05).The gestational age at delivery and neonatal birth weight were significantly lower(P＜0.05), and there was no significant difference in maternal age and neonatal gender (P＞0.05) (Table 2).

Tab2 Comparison of general conditions between the two groups

3.2 The expression of TUSC3 is increased in preeclampsia placentas

Firstly, TUSC3 expression profile was analyzed in the public TUSC3 database GSE12767, which prospectively collected the TUSC3 expression profile in 160 early pregnant women at 10-12 weeks of gestation, including 4 cases with preeclampsia and 6 cases of normal pregnant women.The results showed that TUSC3 expression was increased in the first trimester villi of preeclampsia(Figure 1A).Immunohistochemical analysis of the protein expression levels of TUSC3 in the late placentas of 10 PE and 10 control normal parturient showed that TUSC3 was significantly increased in the late placentas of the PE group, which was consistent with the results of qRT-PCR (Figure 1 B-C).These results indicated that TUSC3 expression was significantly increased in the placenta of the PE group.

Fig 1 TUSC3 Expression in Placental tissues.A.TUSC3 expression in public data GSE12767.TUSC3 expression was compared between PE and normal first trimester villi.In the early pregnancy villi of PE, its expression tended to increase.B.Quantitative real-time PCR was used to detect TUSC3 mRNA expression in 10 PE and 10 normal placentas.The expression of TUSC3 was significantly increased in PE placentas (**P＜0.01).C.Immunohistochemistry was used to detect TUSC3 mRNA expression in 10 cases of PE and 10 cases of normal placentas.TUSC3 expression was significantly increased in PE placentas(**P＜0.01).

3.3 The overexpression of TUSC3 inhibited the migration of HTR8 cells

A scratch assay was used to analyze the migration ability of HTR8 stably overexpressing TUSC3 cell lines and control cell lines (Figure 2).The results showed that the migration rate of TUSC3 overexpression cells was significantly different from that of control cells at 9 hours (P＜0.01), and the migration rate of TUSC3 overexpression cells was significantly decreased.Cell fusion was observed in the control cells at 15 hours, but not in the TUSC3 overexpressing HTR8 cell line (Figure 2B) (P＜0.01).Statistical analysis was performed after the fusion distance was measured,and the migration rate of the two groups was significantly different(Figure 2C).The results suggested that TUSC3 overexpression inhibited the migration of HTR8 cells.

3.4 The overexpression of TUSC3 inhibited the invasion of HTR8 cells

In addition, Transwell assay was used to analyze the invasive ability of HTR8 stably overexpressing TUSC3 cell lines and control cell lines (Figure 3).The results showed that compared with the control cells, the number of cells invading the lower compartment of HTR8 cell lines with overexpression of TUSC3 was significantly reduced(Figure 3A).After statistical analysis of the number of cells invading the lower compartment, there was a significant difference between the two groups (Figure 3.B) (P＜0.01).The results suggested that overexpression of TUSC3 inhibited the invasion of HTR8 cells.

Fig 2 Wound-healing assay.A.qRT-PCR detection of the constructed TUSC3 overexpression HTR8 cell line.B-C.At 9 h, the migration rate of overexpressed TUSC3 cells was significantly different from that of control cells (P＜0.01), and the migration rate of overexpressed TUSC3 cells was significantly decreased.The cells in the control group were fused at 15 hours, while the HTR8 cell line overexpressing TUSC3 did not fuse, and the migration rate decreased significantly (**P＜0.01).The experiment was repeated three times (n=5).

Fig 3 Transwell migration assays.A-B Compared with the control cells, TUSC3 overexpression HTR8 cells significantly reduced cell invasion into the lower chamber, and the cell invasion rate of the two groups was statistically significant (**P＜0.01).The above experiments were repeated three times (n=5).

4.Discussion

PE is a pregnancy-specific disease, and its etiology is closely related to pathological placenta formation[9].Dysfunction of trophoblast migration and invasion impairs the normal function of the placenta and is one of the main mechanisms in the pathogenesis of PE[10,11].The results of this study showed that TUSC3 expression was significantly increased in the placentas of PE patients, and the upregulation of TUSC3 inhibited trophoblast migration and invasion.At the same time, we screened the GSE12767 dataset in the public database, and prospectively collected 160 cases of 10-12 weeks early pregnancy villi, among which 4 cases with PE and 6 cases of normal pregnancy villi were selected for expression profile analysis[12].The results showed that the expression of TUSC3 in the early villi of PE showed an upward trend.We speculate that abnormal TUSC3 expression leads to trophoblast dysfunction, which leads to shallow implantation of placenta and participates in the occurrence and development of PE.The abnormal expression of TUSC3 may affect the function of trophoblast in early pregnancy and participate in the pathogenesis of PE.Therefore, TUSC3 may be a promising marker for early prediction of PE, but further studies are needed.

Trophoblast cells constitute an important part of placental tissue.The main function of trophoblast is to assist embryo implantation and serve as a bridge connecting the embryo and the maternal uterine artery to ensure the nutrient supply of the embryo, which is the basic guarantee for fetal development[13].During the normal first 8-10 weeks of pregnancy, hypoxia (2%) favors the proliferation of trophoblast cells, which anchor blastocysts to the endometrium and block the tips of spiral arteries within the decidua layer of the placenta, limiting blood flow to allow oxygen to enter the intervillous space and promoting trophoblast proliferation.By the 12th week of pregnancy, the placenta increases the oxygen content to 8.5% by establishing continuous low-flow perfusion of oxygenated blood in the intervillous space, which promotes the differentiation of proliferative trophoblast cells into invasive mesenchymal EVTs, and migration and invasion into the uterine wall and spiral artery vascular endothelium, destroying elastin and replacing smooth muscle cells,remodeling spiral artery, and maintaining normal pregnancy[14].Therefore, the differentiation and invasion of trophoblast cells are the basis for ensuring the normal development of placenta and maintaining the progress of pregnancy.

The pathogenesis of PE involves the mother, fetus, placenta and other links.The main theories of etiology and pathogenesis include shallow implantation of placenta, oxidative stress of endothelial cells, imbalance of maternal-fetal immune tolerance, genetic factors.[15].Among them, the more classic is the “two stages” theory.The first stage is the preclinical stage, in the early pregnancy, the trophoblast cells have abnormal differentiation, migration and invasion, which cannot effectively invade the myometrium, leading to the obstacle of “vascular remodeling” of the uterine spiral artery,resulting in “shallow implantation of the placenta”.The second stage is the clinical stage.Due to the obstacle of “vascular remodeling”,placental hypoperfusion, increased oxidative stress, and damaged endothelial cells lead to the release of a large number of placentaderived inflammatory factors into the blood, resulting in a series of clinical symptoms of PE[16].In early pregnancy, trophoblast invasion function is abnormal and cannot fully invade the myometrium,leading to vascular remodeling disorder of the uterine spiral artery and leading to shallow implantation of the placenta, which is the main cause of PE[17].The abnormality of trophoblast invasion function is related to a variety of factors, such as oxidative stress damage of trophoblast cells, endoplasmic reticulum stress, abnormal Epithelial-Mesenchymal Transition (EMT) function, mitochondrial dysfunction, and excessive activation or inhibition of signaling pathways[18].

Trophoblast cells are also a kind of “pseudotumoral” cells, which have the ability of migration and invasion similar to tumor cells.Our previous study found that overexpression of TUSC3 inhibits the migration and invasion of cervical cancer cells by regulating the AKT signaling pathway[8].In small cell lung cancer, downregulation of TUSC3 expression promotes lymph node metastasis, and TUSC3 is significantly higher in node-negative tissues than in normal tissues[19].In hepatocellular carcinoma, overexpression of TUSC3 can inhibit the LIPC/AKT signaling pathway, thereby inhibiting the EMT process of hepatocellular carcinoma cells,leading to the down-regulation of proliferation and invasion ability of hepatocellular carcinoma cells[20].As a “tumor suppressor gene”,TUSC3 participates in the regulation of cell invasion and migration ability.The results of the present study also showed that TUSC3 overexpression significantly reduced the migration and invasion ability of trophoblast cells.Therefore, we hypothesized that TUSC3 might be a new key factor involved in the regulation of trophoblast function.Meanwhile, it has been reported that TUSC3 is involved in the process of N-glycosylation[21].Protein glycosylation occurs in the endoplasmic reticulum of cells, and abnormal glycosylation can destroy the folding of proteins in the endoplasmic reticulum, induce changes in the structure and function of the endoplasmic reticulum,and promote endoplasmic reticulum stress[22,23].Excessive ER stress activates NLRP3 inflammasome through TXNIP pathway and induces pyroptosis to promote the occurrence and development of PE[24,25].Pyroptosis is a pro-inflammatory programmed cell death mode[26], which is characterized by the activation of NLRP3 inflammasome and caspases (mainly caspase-1, 4, 5,11) by risk factors such as PAMPs/DAMPs and ROS.gasdermin family proteins mediated membrane pore formation and increased membrane permeability.Resultsin the rapid release of a large number of inflammatory factors.Recent studies have shown that inflammatory factors such as NLRP3, Caspase-1, IL-1β and IL-18 can be detected in the peripheral blood and placental tissue of PE women, suggesting that the activation of pyroptosis in placental tissue is significantly related to the risk of PE[26].The proportion of pyroptosis in trophoblast cells of PE placenta increases, which causes a large number of inflammatory factors and alarm factors to enter the maternal circulation, inducing sterile inflammation in the pathogenesis of PE and promoting systemic inflammatory cascade[27].Some scholars have found that inhibiting NLRP3 activation in placental trophoblast cells can significantly improve the LPS-induced PE-like symptoms, which further confirms that the up-regulation of NLRP3 in trophoblast cells and the excessive activation of pyroptosis play an important role in the progression of PE[28].Therefore, we speculate that TUSC3 may be involved in the occurrence and development of PE by affecting N-glycosylation to induce ER stress and pyroptosis in trophoblast cells.The molecular mechanism of TUSC3 in regulating trophoblast function still needs to be further studied.

In conclusion, TUSC3 expression was upregulated in PE placentas and inhibited trophoblast migration and invasion.In addition, we also explored the molecular mechanism of TUSC3 regulation, which may provide new ideas and theoretical basis for the prevention and treatment of PE.

Authors’ contribution

Mengyongwei Li and Yanlin Ma: experimental design and paper writing; Lin Dan: Cell experiments; Kang Jinyu: Literature review;Sun Fei and Jie Qiuling: Experimental design and data analysis.

All the authors declare no conflicts of interest.