Correlative Research Of ARRDC3 And Preeclampsia

PART 1:Correlative research of ARRDC3 and preeclampsiaObjective1.To explore the expression of ARRDC3 in the placenta of PE(preeclampsia)and the expression of ARRDC3 in HTR8/SVneo cell under hypoxia.2.To investigate the effect of ARRDC3 on invasion and tube-formation of Extravillous Trophoblast(EVT)cell line HTR8/SVneounder hypoxia.Methods1.Bioinformatic analyses was used to identify gene related to preeclampsia,hypoxia,cell invasion and angiogenesis from four gene expression profiling data sets.Immunohistochemistry was used to detect the expression of ARRDC3 in 10 cases of PE placental tissue and 10 cases of normal placental tissue.Western Blot was used to probe the expression of ARRDC3 in 10 cases of PE placental tissue and 10 cases of normal placental tissue.2.An in vitro hypoxia cell model in extravillous trophoblast cell lines HTR8/SVneo cell was established,Western Blot was used to detected the ARRDC3 expression in HTR8/SVneo cell under hypoxia,EDU assays was used to explore the effect of hypoxia on proliferation of HTR8/SVneo cell,Transwell assays was used to explore the effect of hypoxia on invasion of HTR8/SVneo cell,Tube formation assays was used to explore the effect of hypoxia on angiogenesis of HTR8/SVneo cell,flow cytometry assays was used to explore the effect of hypoxia on apoptosis of HTR8/SVneo cell.3.ARRDC3 DNA recombinant plasmid or mock plasmids were transfected into HTR-8/SVneo cells,Western Blot was used to detect the transfection efficiency,EDU assays was used to explore the effect of ARRDC3 overexpression on proliferation of HTR8/SVneo cell,Transwell assays was used to explore the effect of ARRDC3 overexpression on invasion of HTR8/SVneo cell,Tube formation assays was used to explore the effect of ARRDC3 overexpression on angiogenesis of HTR8/SVneo cell,flow cytometry assays was used to explore the effect of ARRDC3 overexpression on apoptosis of HTR8/SVneo cell.4.SiRNA-ARRDC3 or siRNA-control were transfected into HTR-8/SVneo cells,Western Blot was used to detect the transfection efficiency,EDU assays was used to explore the effect of ARRDC3 silencing on proliferation of HTR8/SVneo cell,Transwell assays was used to explore the effect of ARRDC3 silencing on invasion of HTR8/SVneo cell,Tube formation assays was used to explore the effect of ARRDC3 silencing on angiogenesis of HTR8/SVneo cell,flow cytometry assays was used to explore the effect of ARRDC3 silencing on apoptosis of HTR8/SVneo cell.5.HTR8/SVneo cell was treated with HIF-2 ? inhibitors,Western blot was used to detect the expression of HIF-2 a and ARRDC3 in HTR8/SVneo cell.Results1.By using bioinformatic analyses 277 differentially expressed genes were identified,among them,ARRDC3 was associated with hypoxia,cell invasion and angiogenesis.Immunohistochemical analysis demonstrated that the expression of ARRDC3 protein in the trophoblasts and vasculature.Western blot analysis showed that ARRDC was markedly up-regulatedincreased in PE placental tissue when compare with normal placental tissue.2.1n the model of extravillous trophoblast cell lines HTR8/SVneo cell,Western blot analysis showed that ARRDC was markedly induced by hypoxia,EDU assays analysis showed that hypoxia have no effect on the proliferation of HTR8/SVneo,Transwell assays analysis showed that hypoxia inhibited the invasion of HTR8/SVneo cell.Tube formation assays analysis showed that hypoxia inhibited the angiogenesis of HTR8/SVneo cell.Flow cytometry assays analysis showed that hypoxia promoted the apoptosis of HTR8/SVneo cell.3.The model of ARRDC3 overexpression in HTR8/SVneo cell was constructed,Western blot analysis showed that HTR/8SVneo cells transfected with ARRDC3 DNA recombinant plasmid increased ARRDC3 protein expression by(80.08± 7.32)%,EDU assays analysis showed that ARRDC3 overexpression have no effect on the proliferation of HTR8/SVneo cell,Transwell assays analysis showed that ARRDC3 overexpression inhibied the invasion of HTR8/SVneo cell.Tube formation assays analysis showed that ARRDC3 overexpression inhibied the angiogenesis of HTR8/SVneo cell,flow cytometry assays analysis showed that ARRDC3 overexpression have no effect the apoptosis of HTR8/SVneo cell.4.The model of ARRDC3 silencing in HTR8/SVneo cell was constructed,Western blot analysis showed that HTR/8SVneo cells transfected with siRNA-ARRDC3 decreased ARRDC3 protein expression by(75.12±5.30)%,Transwell assays analysis showed that ARRDC3 silencing promoted the invasion of HTR8/SVneo cell,Tube formation assays analysis showed that ARRDC3 silencing promoted the angiogenesis of HTR8/SVneo cell.5.Western blot analysis showed HIF2 a inhibitor suppresses ARRDC3 and HIF2 a protein expressions.ConclusionOur discovery dentified the up regulation of ARRDC3 in the PE placenta,which induced by hypoxia and inhibited the invasion and angiogenesis of trophoblast.ARRDC3 may associated with the pathogenesis of preeclampsia.PART 2:Research development of preeclampsia and placental spiral artery remodeling failure and maternal vascular factor disorderPreeclampsia is a pregnancy-specific disease and is a leading cause of maternal and fetal morbidity and mortality worldwide.The pathogenesis of preeclampsia is unknown.However,the failure of spiral artery remodeling and imbalance angiogenic factors and anti-angiogenesis factors play an important role in the development of preeclampsia.Angiogenic factors and anti-angiogenic factors in the maternal peripheral circulating blood can be used to predict the onset of preeclampsia and to develop new preeclampsia therapies.