Molecular Mechanism Study Of Activin A-regulated Human Trophoblast Invasion

Background:During physiological embryo implantation and the establishment of the maternal fetal interface,extravillous cytotrophoblasts(EVTs)derived from chorionic villi invade the endometrium and rebuild decidual spiral arteries to provide nutrition and an approximate environment for the fetus,ensuring a successful pregnancy.Trophoblast invasion is the key point in this process and is under accurate control by a complex regulatory network.Mistakes in the regulation of trophoblast invasion could cause serious gestational trophoblastic disease.Insufficient trophoblast invasion is regarded as an important origin of recurrent pregnancy loss and preeclampsia,while uncontrolled invasion is blamed for hydatidiform mole and choriocarcinoma.Activin A,consisting of two βA subunits,belongs to the transforming growth factor-β(TGF-β)superfamily and plays an essential role in the regulation of trophoblast behavior.Our previous studies revealed that activin A promoted trophoblast invasion and the acquisition of endothelial-like tube formation abilities.However,the molecular mechanism underlying activin A-increased trophoblast invasion remains to be further explored,and studies have reported that multiple molecules involved in cell adhesion and cell-extracellular(ECM)adhesion participate in this process.Integrin β3 is a critical molecule in cell adhesion regulation that functions to transmit information between the ECM and cells.Previous studies reported that the expression of integrin β3 was increasingly upregulated in invasive extravillous trophoblasts(iEVTs)and endovascular trophoblasts(eEVTs),suggesting that integrin β3 might be involved in trophoblast infiltration of the endometrium.However,whether integrin β3 participates in activin A-induced trophoblast invasion requires further elucidation.In conclusion,investigating the role of integrin β3 in activin A-mediated trophoblast invasion and the related mechanism is of great significance for understanding pathological pregnancy and clinical prevention and treatment of trophoblast dysfunction-related diseases.Purpose:To explore the role of integrin β3 in activin A-upregulated human trophoblast invasion and underlying signaling pathways.Methods:Based on transcriptome sequencing data of the human immortalized extravillous trophoblast cell line(HTR8/SVneo),we found that the ITGB3 gene,which encodes integrin β3,was significantly upregulated by treatment with activin A for 6 hours.Then,we verified the upregulation of activin A on integrin β3 in HTR8/SVneo cells and EVT at the mRNA and protein levels using RT-qPCR and western blotting,respectively.Subsequently,we treated the cells with human recombinant activin A protein and small interference transfection,combined with functional tests such as Transwell invasion assays and CCK8 proliferation assays,to explore the role of integrin β3 in the process of activin A-mediated trophoblast invasion.Activin A regulates signaling pathways involved in integrin β3 expression.Results:Analysis of transcriptome sequencing data revealed that the differentially expressed genes in the Activin A treatment group in the HTR8/SVneo cell line were enriched in regulating cell adhesion,and the transcription level of ITGB3 was found to be significantly upregulated.We used HTR8/SVneo,primary human EVT,and first trimester human villi explants as in vitro experimental models to further explore the relationship between integrin β3 and activin Ainduced trophoblast invasion.By RT-qPCR and western blot,we confirmed that the mRNA and protein expression of integrin β3 were regulated by activin A.SiRNA-mediated knockdown and Transwell invasion experiments found that knockdown of integrin β3 significantly reduced trophoblast basal invasion and activin A-promoted trophoblast invasion.Furthermore,knockdown of integrin β3 was found to significantly inhibit activin A-mediated EVT outgrowth in villous explants.Subsequently,we used the ALK4/5/7-specific antagonist SB431542 and siRNA-mediated knockdown to explore related signaling pathways in trophoblasts and found that SB431542 pretreatment,ALK4 knockdown and SMAD4 knockdown could all abolish the activin A-upregulated integrin β3 expression.Conclusion:Activin A promotes human trophoblast invasion by upregulating integrin β3 via ALK4SMAD4 signaling.