Mechanisms Of Human Decidual Stromal Cells Derived Extracellular Vesicles Inducing Trophoblast Invasion Through Up-regulation Of N-cadherin

Background and objective:Placenta is the organ which playing a key role in the normal growth and development of fetus.Trophoblast invasion plays an important role in placental formation.Although trophoblast cells showing similar invasive ability of malignant tumor cells,the invasion process of trophoblast cells is closely regulated.It will lead to serious pregnancy-related diseases when trophoblast showing abnormal invasion.The mechanisms of regulating trophoblast cells invasion is complex and has not been fully elucidated which is worthy of our further study.A large number of studies have shown that human decidual stromal cells can secrete a variety of cytokines involving in the regulation of trophoblast invasion,however its specific mechanisms has not been fully understood.Extracellular vesicles(EVs)are a lipid bilayer vesicular body secreted by various cellswhich containing a large number of signal transduction factors.EVs can interact with cells so that regulating their functions and participating in the development of some pathophysiological processes.Studies have shown that EVs play an important role in pregnancy and involved in the occurrence and development of some pregnancy-related diseases like preeclampsia.But the role of extracellular vesicles in pregnancy is still fully unknown.EVs come from a wide range of cells.Uterine decidual stromal cells play an important role in regulating the function of trophoblast cells by secreting a variety of cytokines,but whether uterine decidual stromal cells can also participate in the regulation of trophoblast cells functions by secreting EVs has not been reported so far.In this paper,the effect of human decidual stromal cells derived EVs(HDSC-EVs)on the invasion of trophoblast cells was investigated.We performed experiments to elucidate the molecular signaling regulated by HDSC-EVs.The results could help us to understand the particular mechanisms which regulating trophoblast invasion.Research contents and methods.1.Decidualization of human endothelial stromal cells and Isolation of HDSC-EVs(1)We used MPA and CAMP to induce human endothelial stromal cells decidualization in vitro.(2)Ultracentrifuge ways were used to isolate HDSC-EVs and identified them by transmission electron microscopy and Western blot.(3)The interaction between HDSC-EVs and trophoblast cell line HTR8/SVneo cells was observed by PKH26 extracellular vesicles staining.2.HDSC-EVs induced trophoblast cells invasion by mediating EMT.(1)Transwell assay was used to detect the effect of HDSC-EVs on trophoblast cells invasion.(2)Western blot and RT-qPCR were performed to exam the effect of HDSC-EVs on EMT markers of HTR8/SVneo cells and primary extravillous trophoblast.3.HDSC-EVs promoted trophoblast invasion through Smad2/3-N-cadherin signaling pathway.(1)siRNA was used to knock-down the expression of N-cadherin in HTR8/SVneo cells.(2)Transwell assay was used to exam the effect of HDSC-EVs on the invasion of HTR8/SVneo cells transfected with si-N-cad.(3)Western blot was used to detect the expression of phosphorylated Smad2/3 in HTR8/SVneo cells stimulated by HDSC-EVs.(4)siRNA was used to knock-down the expression of Smad2/3 in HTR8/SVneocells.Then RT-qPCR and Western blot were performed to exam the expression of N-cadherin in HTR8/SVneo cells with or without HDSC-EVsstimulation.(5)High throughput sequencing was used to analyze the composition of miRNAs in extracellular vesicles derived from human endometrial stromal cells and human decidual stromal cells.Result.1.We isolated HDSC-EVs from induced decidual stromal cells.2.HDSC-EVs significantly promoted the invasion of HTR8/SVneo cells(P<0.001).3.RT-qPCR and Western blot showed that HDSC-EVs significantly increased expression of EMT markers of trophoblast cells.4.N-cadherin protein played a key role in promoting the invasion of HTR8/SVneo cells by HDSC-EVs.5.The increase of N-cadherin expression in HTR8/SVneo cells induced by HDSC-EVs was mediated by Smad2/3.6.It was found that many miRNAs increasing in HDSC-EVs which were involved in the regulation of MAPK signaling pathway.MAPK signaling pathway is closely related to cell invasion.Conclusion.1.Human decidual stromal cells can secrete extracellular vesicles and interact with trophoblast cell line-HTR8/SVneo cells.2.HDSC-EVs can significantly promote the invasiveness of HTR8/SVneo cells.3.HDSC-EVs induced the invasion of HTR8/SVneo cells by up-regulating the expression of N-cadherin.4.HDSC-EVs mediated the up-regulation of N-cadherin expression in HTR8/SVneo cells by activating transcription factor Smad2/3.5.High-throughput sequencing showed that HDSC-EVs were rich in miRNAs related to MAPK signaling pathway which could affect cell invasion.