Study Of The Potential Role Of QSOX1 In The Pathogenesis Of Preeclampsia

Objectives:Oxidative stress plays a significant role in the pathogenesis of preeclampsia（PE）,by causing death of trophoblast and consequent dysfunction of placenta.Quiescin sulfhydryl oxidase 1（QSOX1）is upregulated in many types of cancer cell,and it promotes disulfide bonds formation in peptides and proteins as well as hydrogen peroxide（H₂O₂）production.However,the role of QSOX1 in the placenta remains largely unknown.The present study aims to investigate the expression pattern of QSOX1 in PE complicated placentae and the role of QSOX1 in the regulation of trophoblastic function,thus providing in-depth understanding of the putative involvement of QSOX1 in PE development.Methods:From November 2015 to December 2016,Human term placenta from normal（n=31）and PE（n=35）pregnant women who underwent cesarean section in the First Affiliated Hospital of Chongqing Medical University were collected to measure QSOX1 expression and H₂O₂ levels.QSOX1 mRNA level was detected by quantitative real-time polymerase chain reaction（qRT-PCR）.The protein expression of QSOX1was assessed by western blotting and Immunohistochemistry（IHC）.A human H₂O₂ ELISA kit was used to measure H₂O₂ levels in placentas.Down-regulation of QSOX1 in HTR-8/Svneo cells was achieved by siRNA interference.A negative control siRNA（Mock）and 3 QSOX1 siRNAs（siQSOX1）were transfected to HTR-8/SVneo cells,and western blotting was used to analyze QSOX1 and Cleaved caspases protein levels respectively in HTR-8/SVneo cells.Besides,the cell lysate and cell culture medium were collected to determine H₂O₂ levels by using a human H₂O₂ELISA kit.The apoptosis of transfected cells was detected by flow cytometry.In vitro cellular PE model was generated by hypoxic incubation,and the expression levels of QSOX1 and HIF-1αin HTR8/SVneo cells treated with hypoxia were measured by western blotting.Then the HTR-8/SVneo cells were divided into 4 groups as follows:blank control group（Control）,negative control group（Mock）,QSOX1 inhibition group（siQSOX1）andcatalasetreatmentgroup（CAT）.Theprotein expression levels of QSOX1 and Cleaved caspases in these HTR-8/SVneo cells were assessed by western blotting,and apoptosis of HTR-8/SVneo cells was evaluated by TUNEL staining.Results:QSOX1 was overexpressed in the PE placenta.Compared with normal placenta,the level of H₂O₂ in PE placenta was significantly higher.Inhibition of QSOX1 expression in HTR-8/Svneo cells led to decreased protein expression of QSOX1 and Cleaved caspases,and Down-regulating the expression of QSOX1 expression in HTR-8/Svneo cells attenuated cell apoptosis and intracellular H₂O₂ level.Hypoxia significantly promoted QSOX1 and HIF-1αprotein expression in HTR-8/Svneo cells and increased apoptosis of HTR-8/Svneo cells,and knocking-down QSOX1 or hydrogen peroxide treatment rescued hypoxia induced apoptosis of trophoblasts.Conclusion:Hypoxia induced up-regulation of QSOX1 and a consequent elevation in intracellular H₂O₂ increased apoptosis in placentae of pregnancies complicated by PE.