| Objective:Environmental pollution has become an increasingly serious issue with the modern industrialization.Polycyclic aromatic hydrocarbons(PAHs)are produced from incomplete combustion of organic materials and fossil fuel in motor vehicles,residential heating units,power plants,and industries.PAHs are also produced in tobacco smoke,grilling and broiling of food.Benzo[a]pyrene(BaP)belongs to PAHs,which are classified as carcinogenic to humans by the International Agency for Research on Cancer.Exposure to PAHs increases the difficulty of conception and the chance of miscarriage and abnormalities in offspring.Ba P can cross the placenta and thus can be transferred from a mother to her fetus,resulting in fetal and placental developmental toxicology.Placentation is critical in embryo and fetal development.During placental development,trophoblast cellsinvade the maternal decidua and endometrium to remodel spiral arteries.Whether PAHsresult in fetal and placental developmental toxicity throughinhibiting the migration and invasion of human trophoblast is still unknown.This study investigatesthe roles of BPDE,the ultimate carcinogen of BaP,on the migration and invasion of trophoblastcell and their underlying molecular mechanism.Methods:1.The roles of BPDE in HTR8/SVneo cell viability and cellular ROS production were measured by MTTand a ROS assay kit,respectively.2.Cell cycle and apoptosis were analyzed by flow cytometry assays.3.The roles of BPDE in HTR8/SVneo cell migration and invasion were analyzed by wound-healing assay and trans-well assay.4.The effects of BPDE on the interaction of HTR-8/SVneo with HUVEC cells were studied by co-culture and tube formation assays.5.Q-RT-PCR and Western blotting analysis were used to analyze mRNA and protein levels of FAK,SRC,PI3 K,p-PI3 K,AKT,p-AKT,eNOS,MMP2 and Smad2.6.e NOS activity was measured by conversion of L-arginine to NO using nitric-oxide synthase assay kit.7.SC79(activator of AKT)was used to confirm the roles of PI3K/AKT/e NOS passway in BPDE-inhibited migration and invasion of HTR8/SVneo cells.8.The roles of novelmiRNA in BPDE-inhibited migration and invasion of SWan71 cells were investigated by high-throughput sequencing technology and cell biology assays.Results:1.The viability of HTR-8/SVneo cells was gradually decreased with increasing BPDE concentration in a dose-dependent manner;exposure to BPDE greatly increased intracellular ROS in HTR-8/SVneo cells.2.Compared with control,the proportions of cells in the G0/G1 phase significantly increased,whereas those in S phase significantly decreased with increasing BPDE concentrations.Low BPDE concentrations(0.5 and 1.0 μM)did not significantly cause HTR-8/SVneo cell apoptosis,whereas cell apoptosis significantly increased when BPDE concentration was higher than 1.5 μM.At 2.0 μM BPDE,the apoptotic rate reached up 40%.3.After exposure to BPDE,HTR8/SVneomigrated slowly and could not reach the middle area even after 36 h.The higher were BPDE concentrations,the lower were the migration rates.Furthermore,the effect of BPDE on migration was studied using trans-well assay.With increasing BPDE concentrations,the number of migrated cells decreased.Moreover,BPDE significantly reduced the number of HTR8/SVneo cells that have invaded into the membrane relative to control.4.Trophoblast cells were spontaneously interacted with the endothelial cells and the tube-like structures were fingerprinted at 0 and 0.25 μM BPDE.At 0.5-1.0 μM BPDE,endothelial network formation and tube length significantly decreased.Therefore,BPDE can significantly inhibit the angiogenic properties of HUVECs by inhibiting the invasion of HTR8/SVneo cells and their interaction with endothelial cells.5.Compared with control(without treatment with BPDE),protein and mRNA expression levels of FAK,SRC,PI3 K,p-PI3 K,AKT,p-AKT,and eNOS were significantly decreased with increasing BPDE concentrations.Moreover,e NOS activity decreased in a dose-dependent manner.Therefore,BPDE-inhibited migration and invasion was caused by inhibition of FAK/SRC expression and PI3K/AKT/eNOS signaling pathway.6.After treatment with 1 μM BPDE,the protein expression levels of AKT and e NOS significantly decreased and were significantly elevated after further addtion of 10 μM SC79.Furthermore,the compensation effects were also observed in the cell migration and invasion assays.BPDE(1 μM)significantly weakened the abilities of migration and invasion of HTR8/SVneo cells;whereas simultaneous treatment with 10 μM SC79 partly attenuatedthe inhibition effect of BPDE on migration and invasion.The results further confirmed that PI3K/AKT/eNOS signaling pathway was involved in BPDE-inhibited migration and invasion.7.BPDE exposure induced SWan71 cells to express novel mi RNA.Novel miRN-18 and miRNA-193b-3p may participate in the regulation of invasion and migrationof SWan71 cell.Conclusion:We investigated the effects of BPDE on migration and invasion of trophoblast HTR-8/SVneo cellsto gain new insights into the mechanisms of BPDE-inhibited invasion and migration of trophoblasts.BPDE attenuates the expression of FAK/SRC/PI3 K and inactivates the PI3K/AKT/eNOS pathway in HTR8/SVneo cells.Therefore,BPDE inhibits the migration and invasion of trophoblasts by down-regulating the FAK/SRC/PI3K/AKT/e NOS/MMP2 pathway,and BPDE attenuates endometrium vascular remodeling by reduction of trophoblast cell invasion.In addition,BPDE exposure inhibitsmigration and invasion of SWan71 cells,possibly by increasing novel-mi RNA-18 amount and downregulating RAS-GTPase and AKT signaling pathways.This work reveals the mechanism of PAH-inhibited migration and invasion of trophoblast,and provides an experimental evidence to understand the adverse effect of PAH on embryo implantation in early pregnancy. |
