EIF5A1 Promotes Trophoblast Migration And Invasion Via Upregulation Of ARAF

Objective:Trophoblasts dysfunction is one of the pathogenesis of recurrent miscarriage.The normal function of trophoblasts in the maternal-fetal interface is regulated by many factors,which has not yet been elucidated.Research based on pathogenesis is crital for discoverying new treatments.In order to elucidate the role of EIF5A1 gene in the regulation of trophoblast function and the pathogenesis of unexplained recurrent miacarriage(URM),this study conducted clinical sample analysis and a series of cell function assays.Methods:Real-time quantitative PCR and Western blot were used to measure the expression levels of EIF5A1 m RNA and protein in villous tissues of URM and healthy controls(HCs).Immunohistochemistry and Immunofluorescence were performed to study the expression of EIF5A1 protein in the villous tissues of 25 URM and 30 HCs.After overexpression or knockdown of EIF5A1,or inhibition its hypusination by GC7 or site mutation,MTS proliferation,would healing,invasion assay and explant culture model were applied to explore the role of EIF5A1 in regulation of trophoblast proliferation,migration and invasion.Mass spectrometry and bioinformatics methods were used to explore downstream targets and signaling pathways regulated by EIF5A1.The mechanisms of EIF5A1 regulating trophoblasts function was studied by molecular biological methods such as cellular immunofluorescence,Western blot and RNA-binding protein immunoprecipitation.Cell function asssays in vitro were further performed to verify the above regulatory mechanisms.Results:1.The expression level of EIF5A1 in cytotrophoblasts of URM villous tissues was significantly lower than that of HCs.2.Overexpression of EIF5A1promotes trophoblast proliferation,migration and invasion;knockdown of EIF5A1 or blocking its hypusination with GC7 inhibits the above functions;mutation of EIF5A1to EIF5A1_K50A to block hypusination eliminates its regulation of proliferation,migration and invasion.3.Hypusinated EIF5A1 directly binds to ARAF m RNA and promotes ARAF protein expression may via controlling its translation;EIF5A1promotes trophoblast migration and invasion through ARAF-mediated activation of the FAK/ERK signaling pathway.4.The expression level of ARAF protein was decreased in cytotrophoblasts of URM villous tissues,and was positively correlated with the expression of EIF5A1.Conclusions:EIF5A1 promotes trophoblast migration and invasion through ARAF-mediated activation of the FAK/ERK signaling pathway;low levels of EIF5A1 and ARAF may be associated with URM in early pregancy.