Tandem Mass Tag-Based Proteomics Analysis Reveals the Mechanism Underlying the Interleukin-6-Mediated Regulation of Trophoblast Function in Preeclampsia

doi:10.21203/rs.3.rs-1516954/v1

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Tandem Mass Tag-Based Proteomics Analysis Reveals the Mechanism Underlying the Interleukin-6-Mediated Regulation of Trophoblast Function in Preeclampsia

https://doi.org/10.21203/rs.3.rs-1516954/v1

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Objective we investigate the mechanism whereby interleukin-6 (IL-6), an important inflammatory marker, influences trophoblast function during preeclampsia.

Methods Quantitative PCR and enzyme-linked immunosorbent assay were used to determine the IL-6 mRNA and protein levels, respectively. CCK8 and transwell assays were used to detect how IL-6 affects the proliferation and invasion abilities, respectively, of HTR-8/SVneo cells; the tube-forming assay was used to detect how IL-6 affects the angiogenesis ability of human umbilical vein endothelial cells (HUVECs) after their co-culture with HTR-8/SVneo cells. Using tandem mass tag-based proteomics analysis, we screened for different proteins before and after IL-6 stimulation; Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to explore the functions and signal pathways associated with, these proteins.

Results The IL-6 levels were higher in the placenta of preeclampsia group than in normal group. IL-6 suppressed the proliferation and invasion of HTR-8/SVneo cells, but promoted the angiogenesis of HUVECs. Seventy differentially expressed IL-6 downstream proteins were identified; these were enriched with various biological processes, molecular functions, cellular components, and biological pathways.

Conclusions IL-6 regulates trophoblast function by interacting with multiple proteins and pathways. Proteomics-based screening represents a macroscopic means for elucidating the molecular mechanisms underlying preeclampsia.

preeclampsia

IL-6

invasion

proliferation

angiogenesis

TMT proteomics

Preeclampsia, which is an idiopathic disease of pregnancy, has an incidence of about 5–7%. It is the main cause of perinatal morbidity and death of pregnant women and fetuses [1]. An increasing number of studies have shown that preeclampsia is a placenta-mediated pregnancy complication; it is a complex disease involving multiple factors and mechanisms. However, the details regarding its pathogenesis remain unclear [2]. Interleukin 6 (IL-6) is an important protein marker of inflammatory reactions during pregnancy; it is also an important regulator of the immune cascade at the maternal–fetal interface. IL-6 and its receptors (IL-6R and gp130) have been detected in the blood of patients with preeclampsia[3]. The proliferation and invasion of trophoblasts, especially, syncytial trophoblasts, at the maternal–fetal interface are important guarantors for maintaining the immune balance at the maternal-fetal interface. Abnormal trophoblast proliferation, invasion, and angiogenesis lead to the shallow implantation of the placenta at the maternal–fetal interface in early pregnancy, causing pregnancy-associated complications, such as preeclampsia and fetal growth restriction. However, the exact mechanisms underlying the IL-6-mediated regulation of trophoblast function with regard to the development of preeclampsia remain unclear.

In the present study, we aimed to explore the effect of IL-6 on the function of trophoblast cells in preeclampsia. Tandem mass tag (TMT)-labeled protein quantification technology and tandem mass spectrometry analysis were used to detect the differentially expressed proteins in HTR-8/SVneo cell culture supernatants before and after IL-6 stimulation; the role of IL-6 was characterized based on these proteins. Further, to reveal the mechanism whereby IL-6 participates in the occurrence and development of preeclampsia, we combined GO enrichment and KEGG pathway analyses to explore the functions and distribution of, and the signal pathways associated with, these proteins.

2.1 Clinical samples

We included 21 women with single pregnancy whose fetuses were delivered by cesarean section in the obstetrics department of our hospital, including 12 women with preeclampsia (preeclampsia group) and 9 women with normal late pregnancy ( normal group). Then, we collected the clinical data, including general information, regarding these subjects. The diagnostic criteria for preeclampsia were in accordance with the 2018 ISSHP International Practice Recommendations for Hypertension in Pregnancy[4]. None of the women had diabetes, kidney disease, heart disease, or other chronic diseases (except for hypertension in pregnancy), and no acute or chronic infectious diseases. None of the pregnant women showed labor and premature rupture of membranes; further, none of the women had bad habits such as smoking or drinking. This study was approved by the ethics committee of the Fujian Maternity and Child hospital.

2.2 Main cell lines, reagents, and equipment used

The main cell lines, reagents, and equipment used for our study were as follows: HTR-8/SVneo trophoblasts (gifted by Duke University and successfully cultured); human umbilical vein endothelial cells (HUVECs) (Procell); transwell-24 well membrane (Corning); matrigel (base glue; (BD)); cell counting kit-8 (CCK-8) assay kit (Dojindo); crystal violet (Beijing Soleibao Technology Co., Ltd.); 4% paraformaldehyde in DEPC water (Servicebio); fetal bovine serum (PAN biotech); 0.25% trypsin-EDTA (1X) (Gibco); IL-6 (PeproTech); human IL-6 ELISA Kit (Boster); SweScrip RT I First Strand cDNA Synthesis Kit (Servicebio); qPCR Mix (CWBIO); qPCR instrument (CG-02/05, Roche 480, ABI 7500); cell culture plate (Corning); microplate reader (Beijing Pulang New Technology Co., Ltd.); high performance liquid chromatography (nanoUPLC EASYnLC1200, Thermo Scientific, USA); mass spectrometer (Q Exactive HFX, Thermo Scientific, USA); and Chromatography columns: XBridge BEH C18 XP column (15 cm × 2.1 mm, 1.8 µm; Waters, USA) and ReprosilPur 120 C18AQ column (100 µm ID × 15 cm, 1.9 µm; Dr. Maisch, Germany).

2.3 Experimental methods

2.3.1 Quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA)

q-PCR was used to detect the IL-6 mRNA expression in the placental tissues; the expression level of IL-6 proteins in the placental tissue was detected by ELISA. We used the NucleoZol reagent to extract the total RNA. Then, we used the total RNA extracted from placental tissue as transcription template, used cDNA synthesis kit (Servicebio) to reverse transcription to synthesize cDNA, and used a fluorescence qPCR kit (qPCR SYBR® Green Master Mix, CWBIO) to perform the amplification reaction. Relative quantity of cDNA was analyzed by the 2^-ΔΔCt method.The primer design was based on the target sequences in the GenBank database. The forward primer of IL-6 was AAGCCAGGCTGTGCAGATG, and the reverse primer was CTGGCATTTGTGGTTGGGTC. Total IL-6 protein levels in placental tissue were measured using an IL-6 ELISA kit (Boster) in accordance with the manufacturer’s instructions.

2.3.2 CCK8 assay

The effects of IL-6 on the proliferation of HTR-8/SVneo cells were assessed using the CCK-8 assay. After the trypsinization of the cells from each experimental group in the logarithmic growth phase, the cells were resuspended in complete medium to form a cell suspension, and the cell density of the suspension was adjusted to 4×10⁴ cells/ml. The single cell suspension was diluted and added to each well of a 96-well plate (with three replicate wells for each concentration). The plates were incubated in a 37°C incubator for 0, 24, and 48 hours; then, 10% CCK-8 reagent was added, followed by incubation for one hour at 37°C. The OD values of the samples at 450 nm were then measured.

2.3.3 Transwell migration assays

Then, the effects of IL-6 (10 ng/ml) on the invasion ability of HTR-8/SVneo cells were assessed using transwell migration assays. After treatment with IL-6 (10 ng/ml) for 48 hours, the invasion ability of the cells was tested. First, Matrigel was stored in a refrigerator at 4°C until it was completely melted. Then, the Matrigel was diluted with serum-free medium (1:8) on ice. Next, 50 µl of the diluted Martrigel gel was coated onto the upper chamber of transwell. After it solidified completely, 100 µl of the cell suspension from each group (1×10⁵ cells/ml cells) was inoculated in the upper chamber of the transwell. Then, the transwell chamber was placed in a 24-well plate with 10% FBS medium for culture; after culturing for 24 to 48 hours, the transwell chamber was removed and the residual Matrigel glue and cells inside the chamber were wiped using a cotton swab. Next, the chamber was washed thrice with PBS, and the cells that had migrated through the bottom and back of the chamber were fixed with paraformaldehyde and stained with crystal violet; the number of cells was counted under a microscope.

2.3.4 Tube-forming assay

To study the effects of IL-6 on the vascular growth ability of HUVECs, HTR-8/SVneo cells were co-cultured with HUVECs and treated with IL-6 (10 ng/ml) for 48 hours to simulate the angiogenesis environment of the maternal–fetal interface. The average blood vessel density was used to calculate the number of blood vessels in three random microscopic fields. The number of blood vessels in each field was divided by the area of each field of view to obtain the number of blood vessels per square millimeter. The difference of angiogenesis before and after IL-6 treatment and at different concentrations of IL-6 were compared.

2.3.5 Tandem mass tag (TMT)-based proteomics analysis for screening the downstream regulators of IL-6

First, the HTR-8/SVneo cells were stimulated for 48 hours using 10 ng/ml IL-6. The cell culture supernatant was collected before and after stimulation. Then, the proteins were extracted to evaluate the protein quality. The protein samples were subjected to enzymatic hydrolysis using trypsin and TMT labeling. Equal amounts of the labeled peptides were mixed into one pooled aliquot. After desalting treatment, high-pH RP-HPLC was used for fractionation, and 12 different peptides were obtained. Each component was separated by nano-HPLC reversed-phase chromatography and mass spectrometry. The PD/MaxQuant database search software was used for protein identification and quantitative analysis. After the quantitative results were standardized, statistical methods were used to screen for differentially expressed proteins (DEPs). The screening criteria for the DEPs were as follows: P-value (Student's t-test) < 0.05 and fold change ≤ 0.83 or ≥ 1.2. The results of this analysis were visualized with volcano maps and heat maps.

2.3.6 GO and KEGG analyses

The ClusterProliter package was used to perform GO enrichment and KEGG pathway analyses of all the DEP-encoding genes. GO enrichment analysis was used to classify the differentially expressed genes according to the biological process, cell component, and molecular function. A corrected P value (Benjamini Hochberg, BH) < 0.05 was considered statistically significant. KEGG pathway enrichment analysis was used to clarify the functions of the differentially expressed genes and the cell signaling pathways involved; the threshold P < 0.05 was used to obtain statistically significant signaling pathways.

2.4 Statistical analysis

SPSS 25.0 statistical software was used to analyze the data. GraphPad 5.0 was used as the drawing tool. Continuous variables were tested for normal distribution; the mean ± standard deviation was used for the normal distribution and the median [Q25, Q75] was used for representing continuous variables that did not meet the normal distribution. A nonparametric test (Kruskal–Wallis test) was used to analyze the differences between the groups. Differences with P values < 0.05 were considered statistically significant.

3.1 Clinical characteristics of the pregnant women in this study

There was no significant difference between the age and pre-pregnancy BMI of the women from the two groups. The gestational week of delivery in the preeclampsia group was significantly earlier than that of the normal pregnancy group, and the birth weight and placental weight of the newborns were lesser in the preeclampsia group than in the normal pregnancy group (Table 1).

Table 1

Clinical characteristics of pregnant women in this study
	Normal pregnancy	Preeclampsia	P value
Maternal age(years)	32 [29, 35]	31.5[29, 38]	0.858
Pre-pregnancy BMI (kg/m²)	20.95[20.08, 22.43]	22.48[18.42, 25.92]	0.594
Gestational age (weeks)	38.71[38.50, 39.71]	35.92[34.32, 37.54]	<0.001
Systolic BP (mmHg)	119[111,128]	156[146,165]	<0.001
Diastolic BP (mmHg)	69[64,81]	96[91,103]	<0.001
Birthweight (g)	3280[3070,3527.5]	2150[1437.5,3070]	0.004
Placenta weight (g)	625[530,660]	470[375,555]	0.006

3.2 IL-6 mRNA and protein expression levels in placental tissues

The IL-6 mRNA levels of the two groups was detected by qPCR. The difference between the two groups was statistically significant (1.47 ± 1.30 vs. 3.60 ± 1.37, P = 0.018) (Fig. 1A). Next, ELISA was used to detect the protein expression levels of IL-6 in the placental tissues of the two groups. The protein expression level of IL-6 in the preeclampsia group was higher than that in the control group (105.33 ± 42.68 pg/ml vs. 76.87 ± 19.06 pg/ml); the P value was close to 0.05 (P = 0.057) (Fig. 1B).

3.3 The effect of IL-6 on the proliferation of HTR-8/SVneo cells

The CCK-8 assay results showed that the OD values of the samples treated with 0 ng/ml IL-6 at 0, 24, 48, and 72 hours were 0.52 ± 0.02, 0.72 ± 0.04, 1.32 ± 0.07, and 1.67 ± 0.07, respectively; those in the samples treated with 5 ng/ml IL-6 were 0.56 ± 0.02, 0.69 ± 0.02, 1.71 ± 0.08, and 1.41 ± 0.06, respectively; and those in the samples treated with 10 ng/ml IL-6 were 0.55 ± 0.01, 0.65 ± 0.03, 1.01 ± 0.08, and 1.37 ± 0.08, respectively. Compared with the IL-6 0 ng/ml group, the IL-6 5 ng/ml group showed no significant difference with regard to the OD value at 24 h (P = 0.071). However, the OD values at 48 h and 72 h were significantly reduced when the culture was continued (P = 0.034 and 0.002, respectively). The OD values at 24, 48, and 72 h in the 10 ng/ml IL-6 group were significantly lower than those in the 0 ng/ml IL-6 group (P = 0.030, 0.001, and 0.001, respectively) (Fig. 1C).

3.4 The effect of IL-6 on the invasion ability of HTR-8/SVneo cells

The transwell migration assays showed that compared with the cells treated with 0 ng/ml IL-6 (125.00 ± 5.24), the number of cells passing through Matrigel gel in the 10 ng/ml IL-6 group was significantly reduced (100 ± 11.76); this difference was statistically significant (P = 0.002) (Fig. 1D, Fig. 2A).

3.5 The effect of IL-6 on the angiogenesis ability of HUVECs

We co-cultured HTR-8/SVneo cells with HUVECs cell line to simulate the angiogenesis environment of the maternal–fetal interface. The results of tube-forming assays showed that the average blood vessel density of HUVECs treated with IL-6 (10 ng/ml) for 8 hours was lower than that of the control group (25.33 ± 3.79 vs. 16.67 ± 2.52), and the difference was statistically significant (P = 0.030) (Fig. 1E, Fig. 2B).

3.6 TMT proteomics results

3.6.1 Preliminary screening of differentially expressed proteins

After pretreatment, 1345 detected proteins were retained, and a total of 70 DEPs were screened, including two upregulated proteins and 68 downregulated proteins (Fig. 3A, 3B). We visualized the DEPs using a volcano map (Fig. 3C). Looking at the literature, it was found that the genes encoding the DEPs were closely related to preeclampsia[5–17]: DKK1, FLNB, U2AF2, MMP16, ADAMTS13, EIF2S1, AMPD2, LTF, CDH13, C6, PDE5A, TF, CLU (Table 2).

Table 2

DEPs closely related to preeclampsia
Protein name	Gene name	Log_fold change	P value
Q76LX8	ADAMTS13	-0.423	0.004
P26368	U2AF2	-0.376	0.010
O75369	FLNB	-0.397	0.020
P05198	EIF2S1	-0.524	0.008
Q01433	AMPD2	-0.414	0.027
P02788	LTF	-0.484	0.039
P55290	CDH13	-0.324	0.047
P51512	MMP16	-0.457	0.026
P13671	C6	-0.362	0.039
O76074	PDE5A	-0.346	0.035
P02787	TF	-0.289	0.036
P10909	CLU	-0.383	0.030
O94907	DKK1	-0.413	0.008

3.6.2 GO annotation and enrichment analysis

We used GO database for annotation and enrichment analysis, classified 3 types of gene functions according to biological processes, cell components and molecular functions. The results show that the DEPs were mainly associated with biological processes such as post-translational protein modification, negative regulation of canonical Wnt signaling pathway, etc.; biological functions such as low-density lipoprotein particle receptor binding, lipoprotein particle receptor binding, and cadherin binding; and cell components such as focal adhesion, cell-substrate junction, and endoplasmic reticulum lumen (Fig. 4).

3.6.3 KEGG pathway annotation and enrichment analysis

In order to have a more systematic and comprehensive understanding of the biological processes involved in IL-6, the above-mentioned DEP-encoding genes were compared with the KEGG database for differential signaling pathway analysis. Fisher's exact test was also used to analyze and calculate the significance level of each pathway associated with the DEPs. The results showed that the DEPs mainly participated in 141 signal pathways. We set P < 0.05 as the screening criterion and found 11 significantly enriched pathways, including Prion disease, Proteasome, and Wnt signaling pathway (Fig. 5). The pathways closely related to preeclampsia mainly include the Wnt signaling pathway, HIF-1 signaling pathway, MAPK signaling pathway, JAK-STAT signaling pathway, Calcium signaling pathway, cAMP signaling pathway, Ras signaling pathway, etc. (Table 3).

Table 3

Pathways related to preeclampsia in the KEGG enrichment analysis
ID	Map name	Gene	Protein	Count	P value
hsa04310	Wnt signaling pathway	DKK1 PRKACA CACYBP	O94907 P17612 Q9HB71	3	0.044
hsa04066	HIF-1 signaling pathway	TF ELOC	P02787 Q15369	2	0.101
hsa04010	MAPK signaling pathway	FLNB PRKACA	O75369 P17612	2	0.431
hsa04630	JAK-STAT signaling pathway	AOX1	Q06278	1	0.556
hsa04020	Calcium signaling pathway	PRKACA	P17612	1	0.636
hsa04024	cAMP signaling pathway	PRKACA	P17612	1	0.663
hsa04014	Ras signaling pathway	PRKACA	P17612	1	0.689

Trophoblast function is the key to normal placental development and successful pregnancy. After embryo implantation, cytotrophoblasts proliferate and differentiate into syncytial trophoblasts or extravillous trophoblasts (EVTs). Then, EVTs invade the mother's decidua and the uterine spiral artery to reshape the blood vessels to provide enough blood and nutrients for the fetus. Studies have shown that trophoblast proliferation, invasion, and migration and restricted angiogenesis are the key reasons for the occurrence of preeclampsia[1]. We found that IL-6 levels were increased in the placental tissue of the preeclampsia group; we also observed that on the one hand, IL-6 can inhibit the proliferation and invasion of gestational trophoblast cells, and on the other hand, it can promote angiogenesis. which indicates that the mechanisms underlying the role of IL-6 in the pathogenesis of preeclampsia cannot be explained via a single mode. Therefore, we further screened the possible downstream regulators of IL-6 involved in the pathogenesis of preeclampsia through TMT-based proteomics analysis and provided new ideas for perfecting the pathophysiological study of preeclampsia.

IL-6 is a typical cytokine involved in inflammation and immune response. At the same time, IL-6 has a close relationship with pregnancy and preeclampsia. However, there is still controversy about whether the level of IL-6 in preeclampsia is elevated. Some studies have shown that there is no significant difference between the expression of IL-6 in placental tissues during preeclampsia and normal pregnancy[18], and other studies have found that IL-6 level increases [19] or decreases[20] in preeclampsia. However, these studies only revealed the relationship between the IL-6 expression level and preeclampsia and did not thoroughly explore the mechanism whereby IL-6 participates in the pathogenesis of preeclampsia. We found that the IL-6 level was significantly increased in the placental tissue of the preeclampsia group, which is consistent with the results of the study by Ruby Aggarwal et al.[21]. At the same time, our study also found that IL-6 can inhibit the proliferation and invasion of trophoblasts, which mediates the occurrence of disease. Trophoblast proliferation and invasion can lead to insufficient spiral arterial remodeling and insufficient blood perfusion, which are important causes of preeclampsia. Most research reports have shown that IL-6 promotes the proliferation and invasion of tumor cells [22], but it has an inhibitory effect on the invasion of trophoblast cells, which indicates that the mechanism whereby IL-6 inhibits the proliferation and invasion of trophoblast cells is quite different from that in case of tumor cells. The TMT-based proteomics analysis showed that the proteins encoded by FLNB and DKK1 were significantly downregulated in the IL-6 stimulation group in our study. FLNs are networked cytoskeletal proteins connected to the cell membrane surface and play an important role in the integration of cell mechanics and signal transduction. Jufeng Wei et al. found that the mRNA and protein expression levels of FLNB decreased significantly in the placenta tissues during preeclampsia. The downregulation of FLN-b has been reported to inhibit the ERK/MMP-2 and MMP-9 pathways, leading to a reduction in the invasion ability of trophoblasts in the placenta during preeclampsia [6].DKK1 (Dickkopf-1) is a secreted glycoprotein closely related to Wnt signaling. Mariz Kasoha, et al. reported that the expression level of DKK1 decreased significantly in the placenta tissue of patients with preeclampsia, and its decreased level was closely related to the severity of the disease[5]。

At the same time, using KEGG pathway enrichment analysis, we found that the involvement of IL-6 in the pathogenesis of preeclampsia may be related to the Wnt signaling pathway. In the process of placenta formation, there are a variety of signaling pathways involved in regulating the proliferation, differentiation, invasion, and apoptosis of trophoblasts, among which the Wnt signaling pathway is an important one[23]. In addition, the Wnt signaling pathway is also involved in placental implantation. Its excessive activation can lead to trophoblast cell tumors [24]. In recent years, many studies have shown that the Wnt signaling pathway is involved in the invasion of trophoblast cells during pregnancy [25]. Xiaofang Wang et al.[26] detected Wnt1 and β-catenin, the key molecules of the Wnt signaling pathway in placental tissues, and found lower expression of these proteins in preeclampsia patients, further indicating that the Wnt/β-catenin signaling pathway may regulate the invasion and proliferation of trophoblasts, which play an important role in the pathogenesis of preeclampsia.

We co-cultured HTR-8/SVneo cells with HUVECs and found that IL-6 can promote angiogenesis in our study. Preeclampsia is related to superficial placental implantation and oxidative stress induced by ischemia-reperfusion, which leads to increased levels of pro-inflammatory cytokines, excessive inflammatory response, and unbalanced angiogenesis. These changes result in endothelial cell dysfunction and inflammation, which ultimately led to poor maternal and fetal outcomes [27]. IL-6 is a pleiotropic cytokine with pro-inflammatory and anti-inflammatory functions. Its pro-angiogenesis effect has long been reported [28]. However, these findings are inconsistent with the pathophysiological characteristics of angiogenesis inhibition in preeclampsia, which suggests that the participation of IL-6 in the pathogenesis of preeclampsia does not represent a simple negative regulation; its role in promoting angiogenesis may also be one of the factors influencing the protection of trophoblast cell function. The TMT-based proteomics analysis showed that after IL-6 stimulation, the protein encoded by U2AF2 in the supernatant was significantly downregulated. sFLT-1 (soluble Fms-like tyrosine kinase 1) is a negative regulator of angiogenesis; it plays an important role in the pathogenesis of preeclampsia[29]. U2AF2 can splice FLT-1 into sFLT-1, inhibiting angiogenesis, leading to endothelial dysfunction[7].The role of IL-6 in promoting angiogenesis may be related to the downstream pathways inhibiting the expression of U2AF2 and reducing the production of sFLT-1.

In conclusion, we found that IL-6 has a dual role in regulating gestational trophoblast cell function, involving multi-protein and multi-pathway interactions. Further, proteomics screening represents a potential macroscopic measure for studying the molecular mechanisms and prognostic targets associated with the pathogenesis of preeclampsia and subsequently, treatment strategies. However, this study is only an in vitro cytology study; the specific mechanism underlying the participation of IL-6 in preeclampsia needs to be further explored and verified. In future research, we can also establish animal models of preeclampsia to further explore the regulation of trophoblast cell function by IL-6 in vivo and to verify the role of FLNB, DKK1, and U2AF2 and other proteins from different pathways in preeclampsia.

IL-6 has a dual role in regulating the function of trophoblast cells. IL-6 regulates trophoblast function by interacting with multiple proteins and pathways. Proteomics-based screening represents a macroscopic means for elucidating the molecular mechanisms, treatment and prognostic underlying preeclampsia.

Author Contribution Statement

QH, S-SZ, J-YY, and H-LZ conceived and designed the research. S-SZ and QH acquired data and performed the experiment. H-LZ and J-YY performed the statistical analysis. QH and S-SZ drafted the manuscript. J-YY and H-LZ participated in the study design and coordination and helped to refine the manuscript. All authors read and approved the final manuscript.

Acknowledgments

This work was supported by the Fujian Science and Technology Project (2019J01514), Joint Funds for the Innovation of Science and Technology, Fujian province (2020Y9134), Fujian Provincial Health Technology Project (2019-2-10), and Health Research Project of Department of Finance (Fujian finance refers to [2019] No. 827) (2020Y183).

Ethics approval statement

The study was legally approved by the institutional ethics committee of Fujian Provincial Maternity and Children’s Hospital and conducted in accord with the guidelines of the Declaration of Helsinki, and the rights of all participants were protected. All experiments were performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects and/or their legal guardian(s).

Conflict of Interest Statement

The authors have declared no conflict of interest.

Data Statement

Data were available on request from the corresponding author.

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No competing interests reported.

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Tandem Mass Tag-Based Proteomics Analysis Reveals the Mechanism Underlying the Interleukin-6-Mediated Regulation of Trophoblast Function in Preeclampsia

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Abstract

Figures

1 Introduction

2 Methods

2.1 Clinical samples

2.2 Main cell lines, reagents, and equipment used

2.3 Experimental methods

2.3.1 Quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA)

2.3.2 CCK8 assay

2.3.3 Transwell migration assays

2.3.4 Tube-forming assay

2.3.5 Tandem mass tag (TMT)-based proteomics analysis for screening the downstream regulators of IL-6

2.3.6 GO and KEGG analyses

2.4 Statistical analysis

3 Results

3.1 Clinical characteristics of the pregnant women in this study

3.2 IL-6 mRNA and protein expression levels in placental tissues

3.3 The effect of IL-6 on the proliferation of HTR-8/SVneo cells

3.4 The effect of IL-6 on the invasion ability of HTR-8/SVneo cells

3.5 The effect of IL-6 on the angiogenesis ability of HUVECs

3.6 TMT proteomics results

3.6.1 Preliminary screening of differentially expressed proteins

3.6.2 GO annotation and enrichment analysis

3.6.3 KEGG pathway annotation and enrichment analysis

4 Discussion

5 Conclusions

Declarations

References

Additional Declarations

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