| BackgroundSpontaneous abortion(SA)or Miscarriage refers to the abortion before 20 weeks of pregnancy,which is a common reproductive disorders of obstetrics and gynecology.In recent years,with the increase in the incidence of spontaneous abortion,research on spontaneous abortion mechanism is still a hot topic,but the pathogenesis of spontaneous abortion is still unclear.Many studies pointed out the dysfunction of trophoblast invasion easily lead to spontaneous abortion,the maintenance of normal migration and invasion ability of trophoblast cells is an important factor to ensure normal pregnancy.So Extravillous trophoblast cells(EVT)become the tools commonly used to study the pathological mechanism of the spontaneous abortion,and would healing assay and Transwell assay are always used to detect the migration and invasion ability of EVT cells.MicroRNAs(miRNAs)are small non-coding RNAs of about 22 nucleotides in length which may be important in many diseases.By comparing the expression of miRNA-126-3p in the villi of spontaneous abortion and normal early pregnancy,it was discovered that miRNA-126-3p could be related to the occurrence of spontaneous abortion.Total flavonoids from semen cuscutae(TFSC)as a representative Bushenantai drug,how it influences the internal mechanism of EVT cells has not been reported,so the pharmacodynamic mechanism of TFSC on EVT cells has important significance,whether TFSC by regulating miRNA-126-3p to promote the invasion ability of EVT cells is the key in this study.ObjectiveAims to study the miRNA-126-3p expression in chorionic villi in patients with spontaneous abortion and in patients with normal pregnancy,the effect of miRNA-126-3p on invasion and migration function of Extravillous trophoblast cells(EVT);to construct spontaneous abortion state EVT cells model,to explore the pharmacodynamic mechanism of TFSC on the migration and invasion abilities of EVT cells and spontaneous abortion state EVT cells model.Methods1.The role and the function of miRNA-126-3p in spontaneous abortion:to verify the differential expression of miRNA-126-3p in patients with early pregnancy and spontaneous abortion villus tissues by RT-qPCR method,using Lipofectamine(?)2000 as the vector of miRNA-126-3p mimic and inhibitor was transfected into EVT cells,would healing assay and Transwell assay to observe EVT cells’ migration and invasion ability,and RT-qPCR for expression of miRNA-126-3p in EVT cells.2.The effect of TFSC on the migration and invasion abilities of EVT cells:detect the contain of TFSC by UV and HPLC method;MTT method was used to detect TFSC on EVT cell toxicity.Would healing assay and Transwell assay for migration and invasion ability of EVT cells by TFSC treatment.3.The effect of TFSC on the migration and invasion abilities of spontaneous abortion state model EVT cells.by mifepristone treatment:detect the migration ability of EVT with different dosage of mifepristone by would healing assay.By using mifepristone intervention on EVT cells with the transfection of miRNA-126-3p,to construct spontaneous abortion state EVT cells model.The migration ability of the model was tested by would healing assay and the miRNA-126-3p changes of the model was detected by RT-qPCR.With the treatment of TFSC on spontaneous abortion state EVT cells model,would healing assay and Transwell assay were used for different groups of migration and invasion,and RT-qPCR were for the expression of intracellular miRNA-126-3p changes.4.The mechanisms of TFSC promoting the migration and invasion abilities of spontaneous abortion state model EVT cells by mifepristone treatment:using miRanda,miRDB,TargetScan and CLIP four database,to predict target gene of miRNA-126-3p,using GO enrichment analysis and and KEGG pathway analysis to predict cell function and signaling pathway that the target gene of miRNA-126-3p might be involved in.And RT-qPCR and WB were used to verify.Finally,the relationship between miRNA-126-3p and target gene was confirmed by double luciferase reporter gene and chorionic villus tissue.5.TFSC promote the migration and invasion of EVT cells by tarteting MMP9:RT-qPCR was used to verify for the expression of MMP9 mRNA after TFSC treatment.Relevant signaling pathways that TFSC of different dosages and different time points on EVT cells might regulate were tested via western blotting.Results一.The role and the function of miRNA-126-3p in spontaneous abortion1.The expression of miRNA-126-3p in villus of patients with spontaneous abortion was higher than that in normal pregnant women,and there was a significant difference between the two groups(P<0.05).2.Successful transfection of miRNA-126-3p into EVT cells to construct miRNA-126-3p mimic and inhibitor model,compared with normal EVT cells,miRNA-126-3p expression of miRNA-126-3p mimic group was significantly increased(P<0.001),while miRNA-126-3p inhibitor group was significantly lowered(P<0.01).3.Overexpression of miRNA-126-3p group decreased the migration and invasion of EVT cells,cell numbers ware significantly lower(P<0.05);while the migration and invasion of miRNA-126-3p inhibitor group increased,the numbers of cell invasion ware significantly increased(P<0.01).二.The effect of TFSC on the migration and invasion abilities of EVT cells1.The contain of TFSC by UV was 90.876%,HPLC for the detection of TFSC containing concluded 77.75%rutin,quercetin accounted for 0.41%,a very small amount of isorhamnetin for 0.03%,the total content of brass for 78.19%.2.Different dosages of TFSC had no significant toxicity for EVT cells.3.1 μg/ml and 5 μg/ml TFSC could significantly promote the migration of EVT cells by would healing assay,in a time and dose-dependent manner;1μg/ml and 5 u g/ml TFSC could increase the invasion ability;compaired to DMSO group,the invasion cell number of TFSC were statistically significant(P<0.05).三.The effect of TFSC on the migration and invasion abilities of spontaneous abortion state model EVT cells by mifepristone treatment1.30 μmol/L and 60 μmol/L mifepristone significantly inhibited the migration ability of EVT cells,and up-regulated intracellular miRNA-126-3p in EVT cells.2.EVT cells of 30 μmol/L mifepristone intervention after miRNA-126-3p transfection,which was miRNA-126-3p mimic-MIF group,was successfully constructed for spontaneous abortion state EVT cells model.Compared with miRNA-126-3p inhibitor-MIF group,the model had lower migration ability,and higher expression of miRNA-126-3p.3.Compared with miRNA-126-3p mimic negative-DMSO group,the migration ability of miRNA-126-3p mimic-DMSO was decreased.After TFSC treatment,the migration ability of miRNA-126-3p mimic negative-TSZ was better than its DMSO group,and the migration ability of miRNA-126-3p mimic-TSZ was also better than its DMSO group.TFSC could increase the migration of miRNA mimic and inhibitor groups,compared with the control group.4.Compared with miRNA-126-3p inhibitor negative-DMSO group,the migration ability of miRNA-126-3p inhibitor-DMSO was increased.After TFSC treatment,the migration ability of miRNA-126-3p inhibitor negative-TSZ was better than its DMSO group,and the migration ability of miRNA-126-3p inhibitor-TSZ was also better than its DMSO group,and better than miRNA-126-3p inhibitor negative-TSZ.TFSC could increase the migration of miRNA-126-3p inhibitor group.5.The migration ability of miRNA-126-3p inhibitor-TSZ was also better than miRNA-126-3p mimic-TSZ group.TFSC could increase the migration ability of EVT cells,and also promote the migration of spontaneous abortion state EVT cells model.If known down the expression of miRNA-126-3p in EVT cells,the function of TFSC increasing EVT migration ability was more obvious.6.TFSC could increase the invasion ability of the spontaneous abortion state EVT cells model.The invasion cell number of miRNA-126-3p mimic-TSZ was significantly increased than its DMSO group,and had statistical significance,compared with miRNA-126-3p mimic negtive-DMSO group(p<0.05).The invasion cell number of miRNA-126-3p inhibitor-TSZ was significantly increased than its DMSO group,and had statistical significance,compared with miRNA-126-3p inhibitor negative-DMSO group(p<0.05).The invasion cell number of miRNA-126-3p inhibitor-TSZ was increased than miRNA-126-3p mimic-TSZ(p<0.001).7.Compared with miRNA-126-3p mimic negative-DMSO group,the miRNA-126-3p expression of miRNA-126-3p mimic-DMSO and-TSZ group were up-regulated(p<0.05).Compared with miRNA-126-3p inhibitor negative-DMSO group,the miRNA-126-3p expression of miRNA-126-3p inhibitor-TSZ was down-regulated(p<0.05).And the miRNA-126-3p expression of miRNA-126-3p inhibitor-TSZ was down-regulated,compared with miRNA-126-3p mimic-TSZ,the model group(p=0.05).四.The mechanisms of TFSC promoting the migration and invasion abilities of spontaneous abortion state model EVT cells by mifepristone treatment1.Screening of the target genes of miRNA-126-3p target genes through the database,there were ten mRNA about invasion and migration ability of cells,including VEGFA,SDC2,GOLPH3,SLC7A5,PEX5,ITGA6,NAV1,SMURF2,PLXNB2,PPP3CB,about cell angiogenesis function,including CRK,PIK3R2.2.The mRNA level of SLC7A5,NAV1,PLXNB2,PEX5 on miRNA-126-3p mimic were down-regulated,and on miRNA-126-3p inhibitor were up-regulated,by using RT-qPCR.3.After miRNA-126-3p transfection to EVT cells after 48h,PLXNB2 protein levels of miRNA-126-3p mimic group,compared with negative group,was significantly decreased(p<0.01);compared with miRNA-126-3p mimic group,PLXNB2 protein levelof inhibitor group and TFSC group were significantly up-regulated,with statistical difference(p<0.01).4.After miRNA-126-3p transfection to EVT cells after 48h,PEX5 protein levels of miRNA-126-3p mimic group,compared with negative group,was significantly decreased(p<0.05);compared with miRNA-126-3p mimic group,PEX5 protein level of inhibitor group was significantly up-regulated,with statistical difference(p<0.01),but TFSC group without change.5.Dual luciferase reporter gene showed that compared with the wild type PLXNB2-WT+Non-target Control group,the fluorescein expression level of PLXNB2-WT+mimic protein group decreased,with significant difference(p<0.001);while the Non-target mutant PLXNB2-Mut+Non-target Control group had no significant difference compared with PLXNB2-WT+Non-target Control group.The fluorescein expression of PLXNB2-Mut+mimic group,compared with PLXNB2-Mut+Non-target Control group,was a little lower,without statistical difference.PLXNB2 was the target gene of miRNA-126-3p.6.The expression of PLXNB2 in spontaneous villus was decreased than in noamal pregnancy villus by Western blotting.五.TFSC promote the migration and invasion of EVT cells by tarteting MMP91.The expression of MMP9 mRNA in EVT cells was obviously increased after 0.5 μg/ml,1 μg/ml and 5 u g/ml TFSC treatment,MMP9 protein was increased after 1 μg/ml and 5 μg/ml TFSC(p<0.01).The mRNA and protein expression were quite different between 8 h and 12 h TFSC treatment in the dosage of 1 μ g/ml(p<0.01).2.On MAPK signaling pathway,1 μg/ml and 5 u g/ml TFSC up-regulated p-ERK level,without changing total ERK(p<0.05).1 μg/ml TFSC increased p-ERK levels in different time point(1h,2h,8h),especially in 1h,with the most obvious increase in p-ERK expression.TFSC increased p38 phosphorylation in a dose-dependent manner,without changing total p38(p<0.01).3.On AKT signaling pathway,1 μ g/ml and 5 μ g/ml TFSC increased p-AKT(308)in EVT cells but no changes in total AKT expression(p<0.05).5 μ g/ml TFSC increased p-AKT(473)in EVT cells also without changes in total AKT(p<0.05).Treated with 1 μ g/ml TFSC for the indicated times(1,2,4,8,12h),p-AKT(308)and p-AKT(473)were up-regulated in 1h(p<0.05).4.On Notch signaling pathway,compaired with the control group,TFSC highly up-regulated Notchl and Notch2 expression in EVT cells(p<0.01).Treated with 1 μ g/ml TFSC for the indicated times(1,2,4,8,12h),Notch2 was increased in 1h and 8h,especially in 8h(p<0.01),while Notchl not.Conclusion(s)1.The expression of miRNA-126-3p was up-regulated in the spontaneous abortion and the over expression of miRNA-126-3p could inhibit the migration and invasion of EVT cells,and the miRNA-126-3p inhibitor increased the migration and invasion ability.2.Using mifepristone to construct for spontaneous abortion state EVT cells model.30 μmol/L mifepristone significantly inhibited the migration of EVT cells and up-regulated intracellular miRNA-126-3p in EVT cells.The model group had lower migration ability,and higher expression of miRNA-126-3p.3.TFSC could increase the migration and invasion of EVT cells,and activate the signal transduction among MAPK,AKT and Notch signaling pathways by increasing MMP9 expression.4.TFSC treatment on spontaneous abortion state EVT cells model group,decreased the expression of miRNA-126-3p in a certain range,and obviously improved the migration and invasion ability of the model group,in order to show that TFSC could improve the migration and invasion of EVT cells of spontaneous abortion by targeting miRNA-126-3p.5.TFSC could down-regulate miRNA-126-3p expression of spontaneous abortion state model cells,and activate the downstream PLXNB2,so as to promote the migration and invasion of spontaneous abortion state model cells,play an important tole in Bushenantai,pharmacodynamic mechanism on cells to prevent abortion. |
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