The Mechanism Of Exosome MiR-483-5p Regulating The Function Of Endothelial Cells In The Offspring Of Preeclampsia

Embryo-derived cardiovascular disease is a hot topic in the world.Preeclampsia(PE)is one of the most important triggers for cardiovascular disease of embryonic origin.However,the mechanism of embryonic cardiovascular disease caused by preeclampsia is still poorly understood.Preeclampsia is a pregnancy complication that is closely related to maternal health and fetal death.The clinical manifestations of preeclampsia involve multiple systems.The most common clinical symptoms include new-onset hypertension and proteinuria.A large number of epidemiological investigations have found that preeclampsia is associated with increased incidence of cardiovascular and metabolic diseases in the offspring.Exosomes are small vesicles with a diameter of 40-160nm.They are secreted by cells and contain a variety of biomolecules such as DNA,RNA,protein and lipid.Exosomes perform the functions of signal transduction and substance exchange between cells.MicroRNAs(miRNAs)in the exosomes play an important role in signaling.However,the exosomes MicroRNA-mediated regulatory mechanism of offspring cardiovascular function still needs systematic and in-depth study.The study hypothesizes that in preeclampsia,the diseased placenta releases vascular-related regulators which are secreted by exosomes and delivered to the offspring vessels via umbilical cord blood.Thereby,the offspring vasculature releases associated microRNAs inducing offspring to produce corresponding short-term and long-term cardiovascular effects.To this end,this study conducted a deep sequencing analysis of exosome microRNAs in the umbilical vein blood of the preeclampsia and found that miRNA-483-5p was abnormally highly expressed.MiRNA-483-5p may play an important role in regulating the vascular function of the offspring of preeclampsia.In this study,the umbilical vein blood samples of preeclampsia patients and normal pregnant women are used as research objects.The research detects the expression of miRNA-483-5p in the study subjects.Then we constructed a human umbilical vein endothelial cell(HUVEC)line with miRNA-483-5p overexpression,and verifies the transfection effect using qPCR.The effect of changes in miRNA-483-5p expression on biological activities such as proliferation and migration capacity of human umbilical vein endothelial cells is studied by Real-Time Cell Analysis(RTCA).On this basis,bioinformatics software is used to preliminarily predict the target gene regulated by miRNA-483-5p,and Western blot is used to detect the expression level of target gene in human umbilical vein endothelial cells overexpressed with miRNA-483-5p.Part Ⅰ Verification of abnormal expression of miR-483-5p in umbilical vein blood exosomesOBJECTIVEThis part detects the expression of miR-483-5p in the offspring umbilical vein blood exosomes of preeclampsia patients and pregnant women with normal pregnancy.MethodsCollection of umbilical vein blood samples from patients with preeclampsia and pregnant women with normal pregnancies.Exosomes in umbilical vein blood are extracted using exosome extraction kits.Identification of extracted exosomes using nanoparticle tracking analysis techniques,transmission electron microscopy and Western Blot experiments.The miRNA in exosomes is extracted using the miRNA extraction kit,and the expression of miR-483-5p is detected by qPCR in umbilical vein blood of preeclampsia patients and normal pregnant women.ResultsThe exosome extraction and identification results shows that the extracted exosomes are round-like vesicles under transmission electron microscopy;the nanoparticle tracking analysis shows that the diameter of the extracted exosomes is about 100 nm;and Western Blot experimental result shows positive expression of the exosome marker CD63.The qPCR results of miRNA showed that miRNA-483-5p was significantly higher in umbilical vein blood exosomes in patients with preeclampsia than in normal pregnant women.Part Ⅱ Elucidate the role of abnormal expression of miR-483-5p in offspring vascular endothelial dysfunctionObjectiveThis part of the study uses the human umbilical vein endothelium(HUVEC)as a cell line model to overexpress miR-483-5p in endothelial cells,then detects functional changes in proliferation and migration of human umbilical vein endothelial cells.MethodsConstruct a human umbilical vein endothelial cell line with miR-483-5p overexpression using mimic reagents,and verify the transfection effect with qPCR.The effect of changes in miR-483-5p expression on the biological activities of human umbilical vein endothelial cells such as viability,proliferation,and migration capacity were studied by Real-Time Cell Analysis(RTCA).ResultsThe expression of miRNA-483-5p in the human umbilical vein endothelial cell line transfected with mimic reagent is significantly higher than that in the control group(p<0.05).Real-Time Cell Analysis(RTCA)shows that the proliferation and migration of human umbilical vein endothelial cells are significantly decreased after the overexpression of miRNA-483-5p.Part III Predict and validate possible target genes for miR-4835pMethodsMiRNA-483-5p regulated target genes are predicted by using bioinformatics software.The expression level of the target gene in human umbilical vein endothelial cells after the overexpression of miR-483-5p is detected by Western blot.ResultsThe target gene of miR-483-5p that is initially predicted by using bioinformatics software(TargetScan7.2)is G protein-coupled receptor kinase 2(GRK2).Western blot results shows that the expression of GRK2 in human umbilical vein endothelial cells after miR-483-5p overexpression is significantly lower than that in the control group.Conclusion1.The expression of miRNA-483-5p in the umbilical vein blood exosomes of patients with preeclampsia is significantly higher than that of normal pregnant women.2.Overexpression of miRNA-483-5p inhibits the proliferation and migration ability of human umbilical vein endothelial cells.3.G protein-coupled receptor kinase 2 is the target gene of miR-483-5p.Overexpression of miR-483-5p inhibits the expression of GRK2.These results suggest that miRNA-483-5p may play a potential role in microvascular dysfunction in the offspring of preeclampsia.MiRNA-483-5p may affect the vascular function of offspring through targeting regulation of GRK2.