Background
Preeclampsia (PE) is a progressive multisystem illness involving widespread endothelial dysfunction and vasospasm. It usually manifests as hypertension and proteinuria after 20 weeks of gestation or postpartum or hypertension and terminal organ dysfunction with or without proteinuria, with an incidence rate of 5–10% [
1]. Currently, there is no cure for this pregnancy disorder expect for early termination of pregnancy and rapid delivery of the placenta. Therefore, exploring predictive indicators related to the occurrence of PE is of utmost importance for more effective prediction and treatment of PE and for improving maternal and fetal outcomes.
Copper is one of the metal elements necessary for various biological processes, including making energy, connective tissues, and blood vessels [
2,
3]. The fluctuation of copper levels in cells has been associated with the occurrence and development of diseases, including tumor cell proliferation, angiogenesis, and metastasis [
4‐
8]. Several copper-regulating ions have been used in anti-cancer treatment, including copper ion carriers (disulfiram, dithiocarbamate, elesclomol.) and copper chelators (tributyl ether, tetrathiomolybdate.) [
9,
10].
Cuproptosis is a copper-dependent and regulated non-apoptotic death mode that participates in the occurrence and development of many diseases. Unlike apoptosis, scorch death, necrotic apoptosis, and iron-related death, cuproptosis occurs through the direct combination of copper and the fatty acylation component of the tricarboxylic acid cycle (TCA) [
11,
12], which leads to the aggregation of fatty acylated proteins, the loss of iron-sulfur cluster proteins, and protein toxicity stress and cell death. Recent studies have identified several genes related to cuproptosis, which may provide a new strategy for predicting the prognosis of patients with PE [
13,
14].
It was known that cuproptosis was involved in tumor cells’ metabolic programs, such as hepatocellular cancer, lung cancer [
15‐
17]. Besides, cuproptosis-related genes (CRGs) has been verified to regulate the migration and invasion of cells by mediating cell death. Nowadays, the mechanism of PE progression has not been clarified, and obstruction of uterine spiral artery remodeling is the most convincing hypothesis. In this hypothesis, the weakness of trophoblasts’ invasiveness is the key to explain why uterine spiral artery remodeling obstructed.
In the present study, we aimed to comprehensively clarify the molecular alterations and clinical relevance of cuproptosis in the development of PE and verified the role of CRGs in the PE progression by validating common gene profiles in multiple databases and clinical models. Based on the above, we intended to provide potential predictive indicators for the occurrence of PE and lay a foundation for the therapeutic application of cuproptosis regulators in PE.
Methods
Multi-omics data source and preprocessing
RNA sequencing dataset (GSE75010) and corresponding clinical data of patients with PE and non-PE were downloaded from the GEO database (
https://www.ncbi.nlm.nih.gov/geo). This dataset contains gene expression data of 77 normal placental tissues and 80 PE placental tissues. The genes related to cuproptosis were derived from cuproptosis articles. In that article, whole genome CRIPSR/Cas9 positive selection screen using two copper ionophores (Cu-DDC and elesclomol-copper) in OVISE cells. Overlapping hits with FDR score < 0.01 were analyzed [
18]. The R language “limma” software package was used to analyze the gene expression difference between PE patients and normal tissues. In order to obtain more DEGs, | LogFC > 0.1 | and
p < 0.05 were set as thresholds. The visualization of DEG was achieved by building volcano maps, heat maps. Later, the CRGs were used to intersect with DEGs and obtain the DEGs related to cuproptosis between PE patients and normal pregnant women. Also, the differences in expression between healthy pregnant women and PE pregnant women were analyzed.
Gene network and gene enrichment analysis
Gene changes at the genomic level can be identified using microarray technology and bioinformatics analysis. In recent years, bioinformatics methods have been widely used in addition to various other analyses to analyze microarray data to identify DEGs [
19‐
21]. The central genes were identified by protein-protein interaction (PPI) network analysis and ten algorithms of the cytoHubba plug-in. In addition, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) [
16,
22‐
24], and Gene Set Enrichment Analysis (GSEA) were used to determine the potential functions of biomarkers.
Weighted gene co-expression network analysis (WGCNA)
The R language “WGCNA” software package was used to construct a gene co-expression network. First, after clustering the samples according to clinical information, an outlier threshold of 60 was set to screen out outliers. Next, based on the soft threshold parameter β, power function f (x) = x β was used to Convert the Pearson correlation matrix to a weighted adjacency matrix. The appropriate soft threshold was determined. After selecting the power of 6, the weighted adjacency matrix was transformed into a topological overlap matrix (TOM) with topological overlap (TO) based dissimilarity (1-TOM), and clustering was performed through TOM. Finally, genes with similar expression patterns were classified into modules according to the difference in TOM of each gene for average linkage hierarchical clustering. To further analyze each module, the minimum size (genome) of the gene tree was 30. If the distance was < 0.25, the modules were merged. After obtaining co-expressed gene modules, central genes were screened according to the criteria of gene weight (GS) > 0.2, GS p value < 0.05, and module weight (MM) > 0.8 MM p value < 0.05.
Criterion and tissue collection
We collected general information from 40 pregnant women who undergoing obstetric examination at the First Affiliated Hospital of Nanjing Medical University from January 2021 to January 2022 based on inclusion criteria. Inclusion criteria: singleton pregnancy, no hypertension and diabetes before pregnancy, regular prenatal examination, no fetal abnormalities. Then, 40 pregnant women were divided into control group and PE group based on the PE definition of guidelines related to gestational hypertension published by AOCG in 2020 [
25].
Placenta tissues was taken during cesarean section with the patient’s consent before surgery. The placental tissue samples were obtained from the midsection between the chorionic and maternal basal surfaces at different placenta locations, and each sample measured approximately 1 cm × 1 cm × 1 cm. The samples were then washed in PBS buffer and rapidly frozen in liquid nitrogen. Written informed consent was obtained from all patients. This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (registration number: 2018-SR-252).
Real-time fluorescence quantitative (PCR)
Total RNAs were isolated from the placenta or cells in accordance with the standard TRIzol protocol (Life Technologies). RNA (1 μg) was used for cDNA synthesis by using the HiScript III RT SuperMix for qPCR (Vazyme, Nanjing, China) in accordance with the manufacturer’s instructions. qPCR procedure was carried out by using ChamQ SYBR qPCR Master Mix kit (Vazyme, Nanjing, China) in accordance with the manufacturer’s instructions. The abundance of mRNA was quantified using the 2 − ΔΔCT method, which was normalized to the expression level of GAPDH and converted to fold changes, qRT-PCR was performed as previously described [
26].
Statistical analysis
First, we made a descriptive statistical analysis of the PE patients in the database. Shapiro Wilk test was used to determine whether continuous data had a normal distribution. If the data conformed to the normal distribution, a T-test was used to compare the general situation of normal and PE patients and the maternal and fetal outcomes, expressed by mean ± standard deviation. If the data did not conform to the normal distribution, the Kruskal Wallis rank sum test was used to compare the differences between the two groups, expressed by the median value [Q1 quartile - Q3 quartile]. The categorical variables are expressed by frequency and proportion (n [%]). The Chi-square test was used to compare the differences between groups. Benjamin-Hochberg’s method was used to make multiple corrections. FDR < 0.05 indicated a statistically significant standard. Through database analysis, we used linear regression to correlate CRGs with maternal and fetal outcomes of PE. The predictability of biomarkers was analyzed by the receiver operating characteristic (ROC) curve. The p-value was bilateral, and p < 0.05 indicated statistical significance.
Discussion
In the present study, we first analyzed the general situation of normal pregnant women and PE pregnant women in GSE data, finding that the BMI, systolic blood pressure, and diastolic blood pressure were significantly higher in PE pregnant women. Also, the termination of pregnancy occurred earlier in patients with PE compared to healthy pregnant women. On this basis, 2831 DEGs between normal placental tissue and PE placental tissue were screened in the database, including 1722 upregulated and 1109 downregulated genes. Combined with 19 genes related to cuproptosis in the current cancer database, NFE2L2, PDHA1, PDHB, DLD, and GLS showed significant differences between the two groups. Therefore, we speculated that cuproptosis has an important role in regulating the occurrence of PE, indicating the potential role of cuproptosis in predicting PE.
Through hierarchical clustering, adjacency relationship, and heat map analysis, we found that NFE2L2, PDHA1, PDHB, DLD, and GLS have potential roles in predicting and diagnosing PE. In order to clarify that these genes can potentially affect the maternal and fetal outcomes of PE, we assessed the association between these genes and maximum systolic blood pressure, maximum diastolic blood pressure, umbilical artery PI ratio, uterine artery PI ratio, the last week of pregnancy, and neonatal weight of pregnant women. Our results showed that NFE2L2 is the cuproptosis gene most closely related to the occurrence of PE, and its upregulation could significantly improve maternal and fetal outcomes. The other four cuproptosis genes were related to some maternal and fetal outcomes, suggesting that the increased expression of PDHA1, PDHB, DLD, and GLS can improve maternal and fetal prognosis to varying degrees.
Next, ROC curve analysis was used to determine whether the fluctuations in the expression of CRGs could affect the occurrence and development of PE. In order to further determine whether the above four cuproptosis genes could be used as predictive markers of PE for in-depth research, we used qRT-PCR, finding that NFE2L2, PDHA1, PDHB, and DLD, as screened CRGs, could affect the progress of PE by regulating cuproptosis-related mechanisms. However, GLS had no significant difference in the expression of PE placental tissue.
Many studies have proved that cuproptosis mediates cell death by regulating the TCA cycle. In this study, four cuproptosis genes (i.e.,
NFE2L2, PDHA1, PDHB, and
DLD) were found to have different roles in cuproptosis [
27,
28]. The transcription factor encoded by
NFE2L2 - nuclear factor red cell 2 related factor 2 (NRF2) is a key regulator of antioxidants, which maintains homeostasis by detecting the level of oxidative stress in cells. In its inactive form, NRF2 combines with its negative regulator KEAP1. Yet, when reactive oxygen species (ROS) appear, the cysteine residue of KEAP1 is modified, and KEAP2 is separated from NRF2. Then, NRF2 is transferred to the nucleus and acts as a transcription factor in combination with antioxidant response elements. NRF2 is currently recognized as a potential tumor therapeutic target [
29,
30].
PDHA1 and
PDHB are part of pyruvate dehydrogenase (PDH) complexes, a nuclear-encoded mitochondrial multienzyme complex that catalyzes the overall conversion of pyruvate to acetyl coenzyme a and CO2 and provides the main link between glycolysis and the TCA cycle. The PDH complex is composed of multiple copies of three enzyme components: pyruvate dehydrogenase (E1), dihydroceramide acetyltransferase (E2), and thiamine dehydrogenase (E3).
PDHA1 encodes E1 containing E1 active site α and has a crucial role in the function of the PDH complex. PDHB encodes E1 β Yaki [
31,
32]. Dihydrothiamide dehydrogenase (DLD) encodes a class I pyridine nucleotide disulfide oxidoreductase family member.
As a mitochondrial protein,
DLD has an important role in the energy metabolism of eukaryotes; it participates in at least five multi-enzyme complexes and is a necessary component for the complex to complete the reaction. In addition,
DLD, as a flavoprotein oxidoreductase, uses FAD as a cofactor to receive protons and electrons to catalyze the formation of disulfide bonds [
33,
34].
PDHA1,
PDHB and
DLD have an impact on the mitochondrial tricarboxylic acid cycle.
Strengths and limitations
The present study has multiple strengths. First, this is the first study that explored the role of cuproptosis in the occurrence and development of PE. Cell death has always been a focus of interest in cancer research. Many studies have proved that cell death is the basis of tumor occurrence and development [
24,
35‐
37]. Cuproptosis is a new type of cell death, which is different from other cell death mechanisms and mainly depends on mitochondrial respiration. In this study, we screened and compared the database and verified it in placental tissue using qRT-PCR technology. It was found that NFE2L2, PDHA1, PDHB, and DLD, as selected prognostic genes, can affect the progress of PE by regulating cuproptosis-related mechanisms. In addition, the currently recognized hypothesis suggests that the occurrence and development of PE is caused by disorders in uterine spiral artery remodeling. The most important factor among them is usually due to the decreased invasion and migration ability of trophoblasts, so it is crucial to verify the changes in the expression levels of related genes in placenta tissues before further research on the mechanism of cuproptosis can be conducted. In order to further explore the role of copper death related genes in the mechanism of PE and whether it can be used as a serum marker to predict the occurrence of PE is our future research direction.
The present study also has some limitations. First, although these genes were screened through the database and the validation of clinical tissue samples, the number of tissue samples for validation was not large enough. Secondly, the specific regulatory molecular mechanisms of CRG in PE need to be further investigated.
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