The Molecular Mechanism Of Peroxiredoxin 2 In The Regulation Of Trophoblasts Proliferation And Apoptosis At Maternal-fetal Interface

Objective: Recurrent miscarriage occurs in 5% of women of reproductive age.It has complicated etiopathogenesis and is a great challenge in clinical treatment.The objective of our study includes: to investigate the different expression of Peroxiredoxin(Prdx)2 in first-trimester chorionic villous tissues from normal pregnancy and unexplained recurrent miscarriage;to analyze the effect of downregulated Prdx2 in the proliferation and apoptosis of trophoblast cells;to clarify the molecular mechanism of downregulated Prdx2 in the regulation of proliferation and apoptosis;to identify the up-stream transcription factor of Prdx2 and its regulatory mechanism.Our study may further reveal the pathogenesis of recurrent miscarriage and provide new ideas and methods in clinical diagnosis and treatment.Methods: Chorionic villous tissues from patients with unexplained recurrent miscarriage were collected in this study and women that underwent artificial abortion at 6–12 weeks of gestation to terminate normal pregnancies were recruited as health controls.Western blot,quantitative PCR,immunohistochemistry and immunofluorescence were used to study the difference of Prdx2 expression between the two groups;immunohistochemistry and TUNEL assay were applied to detect proliferation marker ki67,apoptotic marker cleaved caspase-3 and DNA strand breaks caused by cell apoptosis.HTR8/SVneo(HTR8)cells were cultured and transfected with small interfering RNA(contrl siRNA and siPrdx2).DCFH-DA fluorescent probes and CCK-8 assay were used to detect cell redox state and cell proliferation with normal or reduced Prdx2 expression.The viability of HTR8 cells transfected with siPrdx2 was tested by CCK-8 kit with different concentration of antioxidant NAC.The influence of Prdx2 knockdown and the use of NAC on cellular proliferation and apoptosis were assessed by BrdU assay and Annexin-V/PI staining.Prdx2 was also knocked down in cultured villous explants and immunofluorescence was used to analyze the expression of ki67.BeWo cells were transfected with control siRNA or siPrdx2 and subsequently treated with FSK.The fusion ratio of BeWo was indicated by E-cadherin rearrangement through immunofluorescence analysis and Syncytin-2 expression was analyzed by quantitative RT-PCR.Western blot was used to detect the changes of p53,phosphorylated p53 and p21 expression in siPrdx2-transfected HTR8 cells with or without antioxidant NAC and p38-MAPK or JNK inhibitor.Placental villous samples were collected to verify the in vitro results by using immunohistochemistry to detect the related molecular changes in placental tissues.A c-Myc binding site was indentified in Prdx2 promoter region using TRANSFAC tool,and ChIP assay was used to verify that binding site.HTR8 cell line was cultured and Western blot,quantitative PCR were used to detect Prdx2 expression with different concentration of c-Myc inhibitor 10058-F4.With the help of siRNA transfection and overexpression plasmid transfection techniques,Western blot and quantitative PCR were used to detect Prdx2 expression with the knockdown and overexpression of c-Myc respectively.c-Myc expression was also measured in placental villi using Western blot,quantitative PCR,immunohistochemistry and immunofluorescence.Correlationship analysis of mRNA expressions between Prdx2 and c-Myc was made.CCK-8,Annexin-V/PI staining and Western blot were applied to detect cell proliferation,apoptosis and p53,phosphorylated p53 and p21 expression when c-Myc was knockdown with siRNA transfection.Results: Prdx2 is downregulated in cytotrophoblasts from patients with unexplained recurrent miscarriage.Prdx2 knockdown upregulated endogenous production of ROS,inhibited trophoblast proliferation,induced apoptosis and damaged cell fusion.The accumulated ROS induced the impairment of cell viability and expression of phosphorylated p53(p-p53)and p38-MAPK/p21.In villous tissues from patients with recurrent miscarriage,lower rate of cell proliferation and higer ratio of cell apoptosis were detected;the expression of p-p53 and p21 were also upregulated.We found a c-Myc-binding site located in Prdx2 promoter.Chromatin immunoprecipitation(ChIP)showed that c-Myc binds directly to the identified region.Suppression and overexpression of c-Myc led to the reduction and increase of Prdx2 expression respectively.c-Myc was downregulated in the first-trimester cytotrophoblasts of patients with recurrent miscarriage.The knockdown of c-Myc inhibited trophoblast proliferation and increased apoptosis.Conclusions: Prdx2 may be involved in the pathogenesis of recurrent miscarriage.Its expression is mediated by c-Myc and it might play an important role in inhibiting trophoblast proliferation,inducing apoptosis and impairing differentiation during early pregnancy,which might have a potential relationship with first-trimester pregnancy failure.