Hypoxia/reoxygenation attenuates migration and invasion of human extracellular villus trophoblast cells via DNA methyltransferase pathway

Objective To explore the regulative mechanism of hypoxia/reoxygen (H/R) for invasion ability of extravillous trophoblast (EVT). Methods Immortalized human EVT cell line HTR-8/SVneo in early pregnancy, primary EVT cells isolated from the villi of placenta in early pregnancy, and villi explants were cultured respectively. A simulation model of H/R environment in EVT cells invading the uterus was established under the conversion conditions of 1% O2 for 24 h to 20% O2 for another 24 h. Western blotting and immunofluorescence assay were used to detect the expression of key factors such as cell invasion, and Transwell chamber test was employed to observe cell migration and invasion. Results After H/R induction, the migration and invasion abilities of HTR-8/SVneo cells were weakened, and the epitaxial ability of cultured villi explants and the migration and invasion abilities of isolated primary EVT cells were also weakened. Under H/R conditions, the expression levels of MMP-2 and MMP-9, integrin-β1 and integrin-α5 were decreased in HTR-8/SVneo cells (P < 0.05), and the protein expression of DNA methyltransferase (DNMTs) was also down-regulated (P < 0.01). In the primary EVT cells under H/R condition, the expression levels of epithelial-mesenchymal transition (EMT) positive regulatory factors, Slug and Snails, were inhibited (P < 0.05), that of epithelial cell marker E-cadherin was increased (P < 0.01), protein levels of MMP-2 and MMP-9, integrin-β1 and integrin-α5 were decreased (P < 0.05), and cell invasion ability was reduced along with decreased DNMT1 and DNMT3A expression (P < 0.05). However, overexpression of DNMT1, DNMT3A and DNMT3B in HTR-8/SVneo cells promoted the up-regulation of interstitial markers N-cadherin, and MMP-2 and MMP-9 (P < 0.05). Conclusion H/R regulates the expression of EMT-related factors such as MMP-2/-9 through DNMTs, and thereby reduces the migration and invasion abilities of EVT cells.