MiR-1291 Participates In The Pathogenesis Of Preeclampsia By Regulating The Function Of Trophoblasts And Endothelial Cells

Part 1:miR-1291 participates in the development of preeclampsia through targeting PGFObjectiveTo explore the correlation between the expression levels of miR-1291/PGF and preeclampsia,and the targeted regulation relationship between miR-1291 and PGF.Methods1.Preeclampsia and normal pregnancy placental tissue were collected.The preeclampsia group included 20 pregnant women who met the diagnostic criteria of preeclampsia,and the control group included 20 healthy pregnant women.2.The expression of PGF in placental tissues was mapped by immunofluorescence assay.RT-PCR,Western blot and immunohistochemical assays were carried out to compare the expression difference of PGF mRNA and protein between PE and normal groups.3.The expression difference of miR-1291 in placenta of the PE and normal groups were detected by RT-PCR assay,and the targeted regulation between miR-1291 and PGF was investigated using dual luciferase reporter gene assay.Results1.The results of immunofluorescence test suggested that PGF was mainly localized in trophoblasts,and some localized in endothelial cells.PGF mRNA and protein expression levels were significantly reduced in placental tissue in preeclampsia compared with the normal group.2.The expression of miR-1291 was significantly increased in the placenta of preeclampsia group.3.The results of dual luciferin reporter gene assay showed that miR-1291 could specifically bind to PGF mRNA 3’UTR and significantly reduce luciferase activity.Conclusion1.Preeclampsia women presented decreasing level of PGF and increasing level of miR-1291 compared with normal pregnant women.2.MiR-1291 can participate in the pathogenesis of preeclampsia through specifically binding PGF mRNA 3’UTR and regulating PGF expression.Part 2:MiR-1291 regulates the biological functions of trophoblasts and endothelial cells through PGFObjectiveTo explore the effects of miR-1291 on the expression of PGF,and investigate the effects of miR-1291 on the proliferation,migration and invasion function of JEG-3 cells,and the effects on the proliferation and tubulation function of HUVEC.Methods1.MiR-1291 mimic/NC and inhibitor/NC were transfected into JEG-3 and HUVEC,and detect the expression level of PGF by RT-PCR and Western blot.2.MiR-1291 mimic/NC and inhibitor/NC were transfected into JEG-3,and CCK8 was used to detect proliferation function,and transwell and invasion experiments were used to detect the migration and invasion functions.3.MiR-1291 mimic/NC and inhibitor/NC were transfected in HUVEC,CCK8 was used to detect cell proliferation function and tube-formation test was used to detect the tubulation function.Results1.The expression of PGF in miR-1291 mimic group was significantly decreased compared with the control group in JEG-3 and HUVEC cells,while the PGF expression was significantly increased in miR-1291 inhibitor group compared with the control group.2.Overexpression or decrease expression of miR-1291 did not significantly change the survival rate of JEG-3 and HUVEC cells at 24h,48h and 72h,and had no significant effect on the proliferation function of trophoblasts and endothelial cell.3.The migration and invasion functions of JEG-3 cells were significantly reduced in the miR-1291 mimic group,and enhanced in the miR-1291 inhibitor group compared with control group.4.The tube formation function of HUVEC was significantly decreased after overexpressing of miR-1291,while the tubule function was significantly enhanced after knockdown of miR-1291.ConclusionBy decreasing the expression of PGF,miR-1291 can inhibit the migration and invasion of trophoblasts,and the tubulation function of endothelial cells.Part 3:Aspirin improves trophoblasts and endothelial cells function by regulating miR-1291/PGF pathwayObjectiveTo explore the regulation of aspirin on the expression of miR-1291/PGF in trophoblast cells and endothelial cells and investigate the effect of aspirin on miR-1291 induced trophoblasts and endothelia cells dysfunction.Methods1.JEG-3 cells were treated with aspirin of different concentrations(0,0.5,1,2mM),and the RT-PCR and Western blotting were carried to detect the expression levels of miR-1291 and PGF.Treated the endothelial cells with ImM aspirin,and detected the expression levels of miR-1291 and PGF by RT-PCR and Western blotting.2.After transfected with miR-1291 mimic,JEG-3 cells were treated with ImM aspirin.Transwell and invasion experiments were used to detect cell migration and invasion functions.3.After transfected with miR-1291 mimic,HUVEC cells were treated with ImM aspirin.Tube-formation assay was used to detect the tubulation function.Results1.JEG-3 were treated with aspirin at concentrations of 0,0.5,1 and 2 mM respectively.Aspirin at different concentrations could inhibit the expression of miR-1291 and promote the expression of PGF.Treatment of HUVEC cells with ImM aspirin also could inhibit the expression of miR-1291 and promote the expression of PGF.Therefore,1mM concentration was selected for subsequent experiments.2.Compared with miR-1291 mimic group,the migration and invasion of trophoblasts were enhanced in the aspirin and miR-1291 mimic group.3.Compared with miR-1291 mimic group,the tubulation function of HUEVC was enhanced in the aspirin and miR-1291 mimic group.ConclusionAspirin can promote the expression of PGF by inhibiting the expression of miR-1291,and improve the trophoblasts and endothelial cells dysfunction induced by miR-1291,which may be one of the mechanisms of preventing the occurrence and development of preeclampsia.