| Objective1-nitropyrene（1-NP）is a nitro polycyclic aromatic hydrocarbon,which is mainly generated by incomplete combustion of fossil fuels and biomass.1-NP exposure during pregnancy is closely related to adverse pregnancy,in which the maternal-fetal interface communication is critical for pregnancy success.In this study,we observed the effects of exosomes derived from 1-NP treated human uterine decidual stromal cells（HDSCs）on trophoblast HTR8/SVneo biological behaviors,and explored the role of Wnt/β-catenin signaling,providing a new idea to elucidating the molecular mechanism of fetal growth restriction induced by 1-NP exposure.Methods1.Decidualization of human endometrial stromal cells and exosomes separation and identification（1）Decidualization of human endometrial stromal cells line was performed in vitro to acquire uterine decidual stromal cells（HDSCs）,and the expressions of m RNA of decidual marker genes PRL and IGFBP1 were measured by q RT-PCR assay.（2）The activity of uterine decidual stromal cells exposed on the different concentrations of 1-NP was evaluate by MTT assay,and consequently determine the exposure concentration of 1-NP.The supernatant of cells after exposure 1-NP was collected,and the exosomes were extracted by ultracentrifugation.（3）The isolated exosomes were identified by transmission electron microscopy（TEM）,nanometry analysis（NAT）,and Western blot.（4）Uptake of exosomes by HTR8/SVneo was assessed by PKH 67 staining assay.2.Role of HDSC-EXOs on biological behaviors of trophoblasts（1）The effects of exosomes on the proliferation,migration,and invasion of HTR8/SVneo cells were observed by plate clone formation assay,scratches assay,and transwell invasion assay.（2）Western blot assay detected the alteration of EMT and Wnt/β-catenin signaling proteins in HTR8/SVneo cells by exosomes from HDSCs.3.Role of Wnt/β-catenin signaling pathway in influencing the biobehavior of HTR8/SVneo cells induced by HDSC-EXOs（1）Wnt/β-catenin signaling was inhibited by DKK-1,and the altered EMT and Wnt/β-catenin signaling proteins in HTR8/SVneo cells were examined by the Western blot assay.（2）Wnt/β-catenin signaling was inhibited by DKK-1,and the effects on the proliferation,migration,and invasion of HTR8/SVneo cells were observed by plate clone formation assay,scratch assay,and transwell invasion assay.Results1.Human endometrial stromal cells were successfully decidualized in vitro and exosomes were obtained and identified.2.The toxic effects of 1-NP on HDSCs were dose-dependent.When the concentration of 1-NP was 10 μM,the survival rate of HDSCs reached 50%.Therefore,the concentration of 1-NP was determined to be 10 μM.3.HDSC-EXOs exosomes can cause biobehavioral changes in HTR8/SVneo cells.The proliferation,migration,and invasion capacity of HTR8/SVneo cells were increased in the exosomes group.Meanwhile,exosomes can promote EMT in HTR8/SVneo cells,Vimentin and N-cadherin is increased significantly,while the expression of E-cadherin is decreased significantly.4.Exosomes can activate the Wnt/β-catenin signaling pathway in HTR8/SVneo cells,and the protein of β-catenin,APC-1,and p-GSK-3β/GSK-3β were significantly increased,with statistically significant differences.5.After DKK-1 treatment,the activation of Wnt/β-catenin signaling pathway by exosomes was inhibited.In addition,HTR8/SVneo proliferation,migration,invasion,and EMT protein expression level were inhibited.Conclusion Exosomes produced by HDSCs after 1-NP exposure can be uptaken by HTR8/SVneo cells,which consequently enhance EMT,proliferation,migration,and invasive behavior.HDSC-EXOs influence cellular biological behavior by activating Wnt/β-catenin signaling pathway in HTR8/SVneo cells.This study clarified the effect of HDSC-EXOs on HTR8/SVneo cells biobehavior from in vitro experiments,and verified the role of Wnt/β-catenin signaling pathway. |
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