Bradykinin Receptor B2R Promotes The Growth And Biological Function Of Human Villous Trophoblastic Cells

objective:This study was to investigate the effect of B2R expression in placenta of PE patients on the function of trophoblast cells.Methods:The level of B2Rprotein expression in placental tissues of early onset severe PE patients(sPE,gestational age less than 34 weeks)were analyzed by Western blot.The effect of B2R on the growth and function of extravillous trophoblast cells was investigated by using HTR-8/SVneo cells.B2R expression plasmid(pcDNA3.1-B2R)was constructed and B2R specific small interfering RNA(si-B2R)was synthesized,then transiently transfected into HTR-8/SVneo cells and pcDNA3.1-CTL and si-CTL were transfected into control group respectively.The expression of B2R in transfected cells was detected by qRT-PCR and Western blot to verify the transfection efficiency of pcDNA3.1-B2R and si-B2R;CCK-8 kits and flow cytometry were used to detect the effects of B2R on the proliferation activity and cell cycle ofextravillous trophoblastcells;and cell scratch test and invasion test detected the effects of B2R on migration and invasion of extravillous trophoblast cells;Cell tube formation assay were used to detect the effects of B2R on vascular capacity of extravillous trophoblast cell and ability of HTR-8/SVneo supernatant to express B2R in human umbilical vein endothelial cells(HUVEC).In addition,qRT-PCR and Western blot were used to detect the mRNA and protein expression of CCND-1 and VEGF-A in the extravillous trophoblast cells after the B2R expression was changed.Furthermore,western blot was used to detect the relationship between the expression of B2R and the signal transduction pathways such as AKT and ERK1/2 in extravillous trophoblast cells.Result:The expression of protein of B2R was significantly down-regulated in placenta tissuesof early onset sPE patients compared with non-infected preterm birth pregnant women.In vitro cell experiments showed that high expression of B2R promoted the proliferation of HTR-8/SVneo cells,and the cell cycle changed from GO/G1 phase to S phase,while low expression of B2R inhibited HTR-8/SVneo cell proliferation activity(P<0.05),and the cell cycle was blocked in G0/G1 phase.In addition,cell scratch assay,invasion assay and cells tube formation assay showed that high expression of B2R significantly increased the migration distance,the number of penetrating cells and tube formation of HTR-8/SV neo cells,while the migration distance,the number of penetrating cells and tube formation of HTR-8/SVneo cells were significantly reduced after the low expression of B2R.In addition,the supernatants of HTR-8/SVneo cells overexpressing B2R promoted HUVEC tube formation.On the contrary,supernatants of HTR-8/SVneo cells with low expression of B2R inhibited HUVEC tube formation.At the same time,we also found that B2R promoted the expression mRNA and protein of CCND-1 and VEGF-Ain HTR-8/SVneo cells.In the molecular mechanism,we found that B2R promotes the expression of VEGF-A by activating the ERK1/2 signaling pathway in HTR-8/Sneo cells,and the expression of VEGF-A is significantly reduced after the suppression of ERK1/2 signaling pathway.Conclusions:The expression of B2R in placental tissues of patients with early onset severe PE was significantly decreased,and in vitro cell test found that B2R had the ability to promote the proliferation,migration,invasion and tube formation of extravillous trophoblast cells.The low expression of B2R in the placenta of PE patients can inhibit the function of extravillous trophoblast cells and is related to the pathogenesis of PE.