J Korean Med Sci.  2023 Apr;38(16):e128. 10.3346/jkms.2023.38.e128.

Differential Expression of NME4 in Trophoblast Stem-Like Cells and Peripheral Blood Mononuclear Cells of Normal Pregnancy and Preeclampsia

• Yu JH
• Jung YJ
• Kim MS
• Cho SR
• Kim YH

• KMID: 2541752
• DOI: http://doi.org/10.3346/jkms.2023.38.e128

Abstract

Background
Preeclampsia (PE) is known to arise from insufficient trophoblast invasion as uterine spiral arteries lack remodeling. A significant reduction in placental perfusion induces an ischemic placental microenvironment due to reduced oxygen delivery to the placenta and fetus, leading to oxidative stress. Mitochondria are involved in the regulation of cellular metabolism and the production of reactive oxygen species (ROS). NME/NM23 nuceloside diphosphate kinase 4 (NME4) gene is known to have the ability to supply nucleotide triphosphate and deoxynucleotide triphosphate for replication and transcription of mitochondria. Our study aimed to investigate changes in NME4 expression in PE using trophoblast stem-like cells (TSLCs) from induced pluripotent stem cells (iPSCs) as a model of early pregnancy and peripheral blood mononuclear cells (PBMNCs) as a model of late preterm pregnancy.
Methods
Transcriptome analysis using TSLCs was performed to identify the candidate gene associated with the possible pathophysiology of PE. Then, the expression of NME4 associated with mitochondrial function, p53 associated with cell death, and thioredoxin (TRX) linked to ROS were investigated through qRT-PCR, western blotting and deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay.
Results
In patients with PE, NME4 was significantly downregulated in TSLCs but upregulated in PBMNCs. p53 was shown to be upregulated in TSLCs and PBMNCs of PE. In addition, western blot analysis confirmed that TRX expression had the tendency to increase in TSLCs of PE. Similarly, TUNEL analysis confirmed that the dead cells were higher in PE than in normal pregnancy.
Conclusion
Our study showed that the expression of the NME4 differed between models of early and late preterm pregnancy of PE, and suggests that this expression pattern may be a potential biomarker for early diagnosis of PE.

Keyword

Induced Pluripotent Stem Cells; Trophoblast Stem-Like Cells; Transcriptome Analysis; Preeclampsia; Cell Death; Mitochondrial Matrix

Figure

• 

  Fig. 1 Generation and characterization of iPSCs derived hAECs. (A) The morphology of iPSCs derived from hAECs of normal and PE was confirmed. (B) ALP staining was used to characterize iPSCs from normal and preeclampsia patients (scale bar = 100 μm). (C) To confirm the characteristics of iPSCs, the expression of OCT4 and SSEA-4, which are human ESC-specific markers, were confirmed by immunocytochemistry. (D) In order to confirm the pluripotency of iPSCs, the expression of markers representing ectoderm (Pax6 or Nestin), mesoderm (Brachuyury), and endoderm (AFP) were confirmed by immunocytochemistry. Each marker was merged with DAPI (scale bar = 50 μm).iPSC = induced pluripotent stem cell, hAEC = human amniotic epithelial cell, PE = preeclampsia, ALP = alkaline phosphatase, ESC = embryonic stem cell, AFP = α-fetoprotein, DAPI = 4,6-diamidino-2-phenylin-dole.

• 

  Fig. 2 Differentiation and characterization of TSLCs derived iPSCs. (A) The change of morphology in TSLCs s through BMP4 treatment was confirmed (scale bar = 50 μm), (B) western blotting confirmed that the expression of CK7, human trophoblast marker, was significantly increased in both the normal and PE groups after BMP4 treatment. Results are expressed as means ± SEM from three independent experiments.TSLC = trophoblast stem-like cell, iPSC = induced pluripotent stem cell, BMP4 = bone morphogenic protein 4, CK7 = cytokeratin 7, PE = preeclampsia, SEM = standard error of the mean.*P < 0.05 versus normal group.

• 

  Fig. 3 Cell death-related expression of p53 upregulated in PE. (A) Western blotting result showed that the expression of p53 was significantly increased in TSLCs (B) qRT-PCR result showed that the expression of p53 was significantly increased in PBMNCs of PE (C) Clinical TUNEL staining was employed to evaluate the level of cell death (green, TUNEL, DAPI nuclear staining) in TSLCs. TSLCs of PE group exhibited increased number of TUNEL positive rate which was normalized to the normal group (scale bar = 20 μm) (D) Western blotting result showed that the expression of TRX tend to increase in TSLCs of PE. Results are expressed as means ± SEM from three independent experiments.PE = preeclampsia, TSLC = trophoblast stem-like cell, qRT-PCR = quantitative reverse transcription-polymerase chain reaction, PBMNC = peripheral blood mononuclear cell, TUNEL = deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling, DAPI = 4,6-diamidino-2-phenylin-dole, TRX = thioredoxin, SEM = standard error of the mean.*P < 0.05 versus normal group.

• 

  Fig. 4 NME4 expression decreased in a model of early pregnancy and increased in a model of late preterm pregnancy of PE. (A) qRT-PCR results showed that the expression of NME4 was significantly decreased in TSLCs. (B) western blotting result showed that the expression of NME4 was significantly decreased in TSLCs. (C) qRT-PCR result showed that the expression of NME4 was significantly increased in PBMNCs of PE. Results are expressed as means ± SEM from three independent experiments. NME4 = NME/NM23 nuceloside diphosphate kinase 4, PE = preeclampsia, qRT-PCR = quantitative reverse transcription-polymerase chain reaction, TSLC = trophoblast stem-like cell, PBMNC = peripheral blood mononuclear cell, SEM = standard error of the mean.*P < 0.05, **P < 0.01, ***P < 0.001 versus normal group.

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