MicroRNA-486-5p Is Involved In The Pathogenesis Of Pre-eclampsia Via Regulation Of NOTCH2

Objective: The study established the differential expression profile of microRNA(miRNA)in placenta of patients with Preeclampsia(PE)and the difference was verified by placental tissue,plasma and mononuclear cells.In addition,this study also explored the clinical value of differentially expressed miR-486-5p as a biomarker for the diagnosis of PE and its involvement in the pathogenesis of PE through NOTCH2.Methods:(1)RNA-sequencing(RNA-seq)technique was used to analyze the differentially expressed miRNAs in placenta of 5 patients with PE and 5 normal pregnant women;(2)Real time fluorescence quantitative PCR(RT-qPCR)was used to analyze the expression level of miR-486-5p in placental tissue,mononuclear cells and plasma of all samples and the correlation between the expression level of miR-486-5p in plasma and clinical indexes was analyzed.(3)NC,miR-486-5p mimic,inhibitor NC and miR-486-5p inhibitor were transiently transfected with lipo3000 into trophoblast cell lines JEG-3 and BeWo cells and vascular endothelial cell line HUVECs.(4)The effects of miR-486-5p on human trophoblast cell lines BeWo and JEG-3 were investigated by cell proliferation,transwell and scratch experiments in vitro.(5)Enzyme linked immunosorbent assay(ELISA)was used to explore the effect of miR-486-5p on inflammatory factors,interleukin-6(IL-6)and tumor necrosis factor α(TNF-α)in vascular endothelial cell line HUVECs.(6)The online databases Targetscan,miRDB and miRWalk predict the miR-486-5p targeted genes and the target gene interaction map is drawn by using STRING online website.Then,the double fluorescence experiment was used to detect whether miR-486-5p could combine with the specific sequence of NOTCH2 to confirm its direct targeting effect.(7)The expression of NOTCH2 protein was detected by Western blotting(WB),and the correlation between miR-486-5p and NOTCH2 in placental tissue was analyzed.(8)The expression of NOTCH2 mRNA was detected 24 hours after transfection of NC,miR-486-5p mimic,inhibitor NC and miR-486-5p inhibitor into trophoblast cell lines BeWo and JEG-3 cells by RT-qPCR and the expression of NOTCH2 protein 48 hours after transfection was detected by WB.(9)The different changes of trophoblast cell line BeWo transfected with miR-486-5p inhibitor + si-NOTCH2,miR-486-5p inhibitor and inhibitor NC were investigated by cell proliferation and transwell experiments in vitro.Results:(1)The results of RNA-seq showed that there were 72 differentially expressed miRNAs in the placental tissue of PE patients,of which 34 miRNAs showed upward trend and 38 miRNAs showed downward trend(2)RT-qPCR showed that the expression level of miR-486-5p was up-regulated in placental tissue,mononuclear cells and plasma in PE.Among them,whether in plasma or mononuclear cell samples,the expression level of miR-486-5p in patients with severe PE(s PE)and EOPE increased more significantly.The expression level of miR-486-5p showed a significant linear positive correlation with systolic blood pressure(SBP),diastolic blood pressure(DBP),mean arterial pressure(MAP),serum creatine(Scr),Alanine transaminase(ALT)and Aspertate Aminotransferase(AST)(p<0.05).(3)In vitro,compared with NC,miR-486-5p mimic restrained the migration,invasion and proliferation of BeWo cells and JEG-3 cells.Compared with inhibitor NC,the ability of migration,invasion and proliferation was enhanced after transfection of miR-486-5p inhibitor in BeWo and JEG-3 cells.(4)ELISA results showed that IL-6 and TNF-α in HUVECs cells transfected with miR-486-5p mimic were higher than those in NC group.Similarly,compared with the inhibitor NC group,the inflammatory factors of HUVECs cells transfected with miR-486-5p mimic decreased significantly.(5)The correlation between miR-486-5p and NOTCH2 showed that the expression level of NOTCH2 in placenta of patients with PE was significantly downregulated compared with normal pregnant women.Meanwhile,RT-qPCR results and pearson correlation analysis showed that the expression of miR-486-5p in placental was negatively correlated with the expression of NOTCH2.(6)Validation of miR-486-5p inhibiting NOTCH2 expression: In BeWo,JEG-3 and HUVECs cells,the level of NOTCH2 mRNA was significantly down-regulated after overexpression of miR-486-5p,and significantly up-regulated after inhibition of miR-486-5p.In addition,after overexpression of miR-486-5p in BeWo and JEG-3 cells,the level of NOTCH2 protein decreased significantly.While inhibiting miR-486-5p in trophoblast cells,the expression level of NOTCH2 protein increased significantly.In order to confirm the direct targeting effect of miR-486-5p and NOTCH2,the results of double fluorescence experiment showed that it was down-regulated when BeWo cells were co-transfected with wild-type vector,but there was no change when co-transfected with mutant vector,which confirmed that miR-486-5p could directly target NOTCH2(7)Compared with inhibitor NC group,inhibition of miR-486-5p could promote the proliferation 、 migration and invasion of BeWo cell,while knockdown of NOTCH2 after inhibition of miR-486-5p could inhibit the proliferation and migration of BeWo cell.Conclusions: Compared with normal pregnant women,miR-486-5p was highly expressed in placental tissue,mononuclear cells and plasma of PE,whereas NOTCH2 was poorly expressed in placental tissue of PE.The functional analysis of miR-486-5p and NOTCH2 showed that miR-486-5p could limit the proliferation,invasion and migration of trophoblast cells and enhance the expression of inflammatory factors in vascular endothelial cells,while NOTCH2 can reverse the functional effect caused by miR-486-5p.What’s more,the expression of NOTCH2 mRNA and protein were down-regulated after overexpression of miR-486-5p.In general,miR-486-5p may be a diagnostic marker of PE and participate in the occurrence and development of PE through miR-486-5p / NOTCH2 axis..