Identification of transcription factors that regulate placental sFLT1 expression

Soluble FMS-like tyrosine kinase 1 (sFLT1) is a decoy receptor of vascular endothelial growth factor. Increased sFLT1 levels have been associated with preeclampsia, chronic kidney diseases, and kidney transplant rejection. However, lower levels of sFLT1 also exhibit beneficial properties in various conditions, such as organization of actin cytoskeleton in podocytes, and immune regulation in healthy pregnancy. Therefore, understanding the transcriptional regulation of sFlt-1 and preserving appropriate expression levels are critical for effective treatment of preeclampsia and other diseases. Cytotrophoblasts (CTBs) were isolated from three first trimester placentas and differentiated into extravillous trophoblasts (EVTs) for six days. RNA was extracted at different time points and used for RNA sequencing. Differentially expressed genes (DEGs) and transcription factors (DETFs) were analyzed. TF enrichment analysis and pathway analysis were performed on DEGs screened from EVTs and CTBs. TF inhibitors were added to primary CTBs directly or during CTB to EVT differentiation to confirm the regulatory effect of TFs on sFLT1 expression. In total, 197 transcription factors were differentially expressed between CTBs and EVTs, among which, 15 DETFs (EPAS1, ETS1, TBX3, CEBPB, FLI1, TEAD4, GATA4, TBX2, LMX1B, ARNT, FOXM1, ERF, PRDM1, TFAP2A and NR2F2) that potentially regulate sFLT1 expression were predicted by ChEA3 and KnockTF software. The mRNA levels of 15 DETFs were validated upon CTBs differentiation into both EVTs and syncytiotrophoblast. The regulatory effects of FOXM1 and CEBPB were confirmed in vitro experiments, and their expression patterns were validated during CTBs differentiation into EVTs and in first trimester placentas. Pathway analysis showed that sFLT1 was involved in P13K-Akt, Rap1, MAPK, Ras and HIF-1 signaling pathway, focal adhesion, and cytokine-cytokine receptor interaction. Protein-protein interaction analysis showed FLT4, PDGFB, TGFB1, IL6R, TNFRSF1B, CSF1R, TGFB2 to be interacted with FLT1. The identified transcription factors can serve as therapeutical targets in preeclampsia to keep the sFLT1 levels within appropriate limits. Overall design: CTBs were isolated from first trimester placentas of three donors and differentiated into EVTs for six days. RNA was collected on day 0, day 3 and day 6 accordingly, and used for RNA sequencing.