Hypoxia/reoxygenation Impairs Migration And Invasion Of Extravillous Trophoblast Cells Involving Down-regulates DNMTs And Reversed EMT

Introduction: Successful pregnancy needs invasion of extravillous trophoblast cells(EVT)into the maternal uterine decidua and vasculature.The environmental factor hypoxia-reoxygenation(H/R)has been emerged to contribute to placental pathology like preeclampsia(PE)with deficient EVT invasion,but its mechanism is not well understood.Methods: Cell viability,apoptosis and cell cycle were analyzed by Flow cytometry(FCM).Western blot was used to test the expression of PCNA,DNMTs,MMP-2 and-9,TIMP3 integrin-1 and-5 and EMT factors.Immunofluorescence staining(IFC)was used to identify the purity of primary EVT.Cell migration and invasion abilities were measured by Transwell assay.Villous explant outgrowth was evaluated to confirm the migration of primary EVT.Results: Cell viability,apoptosis and cell cycle were not affected by H/R.DNMT1 and DNMT3 a were down-regulated in both cells,while DNMT3 b was reduced only in HTR-8/SVneo cells.Expressions of EMT promoting factors were decreased and the epithelial factor E-cadherin was increased in primary EVT,while they remained unchanged in HTR-8/SVneo cells.The migration and invasion abilities of primary EVTs and HTR-8/SVneo cells,as well as the explant outgrowth,were reduced after H/R treatment.Reduced protein levels of invasion relative factors MMP-2 and-9,as well as integrin-1 and-5,were observed in both primary EVT and HTR-8/SVneo cells.Conclusions: H/R impaired EVT migration and invasion was associated with down-regulation of MMP-2 and-9 as well as integrin-1 and-5 involving decreased DNMTs expressions and reversed EMT process.The present study provides insights into the mechanism of pregnant disease with insufficient invasion like PE.