Single-cell sequencing of trophoblasts in preeclampsia and chemical hypoxia in BeWo b30 cells reveals EBI3, COL17A1, miR-27a-5p and miR-193b-5p as hypoxia-response markers
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Background
Preeclampsia (PE) complicates 2–8% of pregnancies and is marked by placental hypoxia and HIF-pathway activation, especially in early-onset PE (eoPE). Integrating patient tissue analyses with experimental models may reveal common molecular markers of trophoblast hypoxic response.
Methods
We analyzed scRNA-seq data from 10 eoPE, 7 late-onset PE (loPE), and corresponding control placentas, identifying villous cytotrophoblast (VCT), syncytiotrophoblast (SCT), and extravillous trophoblast (EVT) subpopulations. BeWo b30 cells were treated for 24 h with CoCl 2 (300 µM) or an oxyquinoline derivative (OD, 5 µM) to induce hypoxia. RNA and small RNA sequencing quantified mRNA and microRNA changes. PROGENy inferred pathway activities.
Results
Single-cell analysis revealed highest hypoxia pathway activation in eoPE, with EVT showing maximum activity among trophoblast populations. Nine genes were upregulated across all trophoblast types in eoPE: EBI3, CST6, FN1, RFK, COL17A1, LDHA, PKP2, RPS4Y1 , and RPS26. In vitro , OD induced more specific hypoxia responses than CoCl 2 , with 1,284 versus 3,032 differentially expressed genes respectively. Critically, EBI3, FN1 , and COL17A1 showed concordant upregulation in both placental tissue and OD-treated cells, while CoCl 2 treatment produced opposite expression patterns. MicroRNA analysis identified hsa-miR-27a-5p and hsa-miR-193b-5p as consistently elevated in both experimental conditions and previously reported in PE placental vesicles. We also identified isoforms of hsa-miR-9-5p and hsa-miR-92b-3p as hypoxia-associated in trophoblast.
Conclusions
EBI3, COL17A1 , hsa-miR-27a-5p, and hsa-miR-193b-5p emerge as trophoblast hypoxia markers in PE. Oxyquinoline derivatives offer a more physiologically relevant in vitro hypoxia model than CoCl 2 . This integrated approach advances understanding of PE pathophysiology and suggests candidate therapeutic targets.
