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

Abstract: 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 hypoxia. 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 CoCl2 (300 $\mu$M) or an oxyquinoline derivative (OD, 5 $\mu$M) to induce hypoxia. RNA and small RNA sequencing quantified mRNA and microRNA changes. PROGENy inferred pathway activities. Results. ScRNA-seq 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 CoCl2, 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 CoCl2 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 CoCl2. This integrated approach advances understanding of PE pathophysiology and suggests candidate therapeutic targets.