| Objective:Fetal growth restriction(FGR),also known as intrauterine growth restriction(IUGR),refers to the failure of a fetus to grow as well as it should in utero.This disease is liable to cause fetal asphyxia or fetal death in utero during pregnancy and affects the long-term health of such fetuses after adulthood.In this study,we isolated and identified exosomes from cord blood of growth-restricted fetuses and used sequencing technology and bioinformatics analysis to explore the differences of microRNAs in cord blood exosomes between growth-restricted fetuses and normal fetuses.To further investigate the effects of miR-33b-5p and its target gene IGF2BP1 in umbilical blood exosomes on biological behavior of umbilical vein endothelial cells in growth-restricted fetuses and the related regulatory mechanisms.Methods:1.Clinical data,umbilical venous blood and umbilical cord samples of growth restricted fetuses were collected.In order to reduce the interference of environmental factors and other factors,we selected the cases of bichorionic biamniotic sac twins with inconsistent growth(two separate placentas without blood flow traffic)as the study object.To further verify the reliability of the results,singleton samples were collected for control.2.Hematoxylin-eosin staining was performed on the umbilical cord to compare the differences in vascular diameter and cross-sectional area of umbilical veins and umbilical arteries between growth restricted fetuses and normal fetuses.3.Exosomes in umbilical cord blood were extracted by overspeed centrifugation,and the presence of exosomes in umbilical cord blood was identified by transmission electron microscopy,nanoparticle tracking analysis and Western blot.4.Nanoparticle tracking analysis was used to detect whether there were differences in the total amount of exosomes between growth-restricted fetuses and normal fetuses,and Western blot was used to verify whether there were differences in the expression levels of exosomal surface mask proteins.5.Cell culture technology was used to culture Human umbilical vein endothelial cells(HUVEC),umbilical blood exosomes were added into the culture environment,and exosome tracer method was used to confirm that HUVEC could phagocytic umbilical blood exosomes.Cell Counting Kit-8(CCK-8),scratch and angiogenesis experiments were used to detect HUVEC proliferation,migration and angiogenesis ability of umbilical cord blood exosomes added in different groups.6.Umbilical cord blood exosomes of growth-restricted fetuses and normal fetuses in twins were selected for miRNA sequencing analysis,and cluster analysis such as GO and KEGG was performed for differentially expressed miRNAs,and it was found that there were differences in the expression levels of miR-33b-5p.The expression of miR-33b-5p in cord blood exosomes between growth-restricted and normal fetuses was verified by Real-time PCR.HUVEC stable cell lines with miR-33b-5p overexpression and silence were constructed,and their proliferative,migratory and angiogenic abilities were detected by CCK-8,scratch and angiogenesis assay.7.Bioinformatics software was used to predict the target genes binding to miR-33b-5p,and it was found that there was a correlation between IGF2BP1 and miR-33b-5p.Dual luciferase reporter gene was used to detect the targeted binding effect and binding site of IGF2BP1 and miR-33b-5p.The differential expression of IGF2BP1 in umbilical blood exosomes of growth restricted fetuses and normal fetuses was verified by Real-time PCR.8.Construct a stable cell line with co-overexpression of miR-33b-5p and IGF2BP1,and conduct proliferation,migration and angiogenesis detection.Western blot was used to detect the effect of differential expression on PI3K/AKT pathway.Results:1.Birth weight,neonatal head circumference,neonatal abdominal circumference and body length were lower in the growth restricted fetal group,and there were significant differences between the two groups(P < 0.05).In terms of blood vessel diameter and cross-sectional area,both umbilical vein and umbilical artery were significantly decreased in the growth restricted fetal group compared with the normal fetal group(P < 0.05).2.After ultra-fast centrifugation,the presence of exosomes in umbilical cord blood was confirmed by transmission electron microscopy,nanoparticle tracking analysis and Western blot.Under the premise of the same serum level,there was no significant difference in the expression level of exosome specific membrane proteins between the growth restricted fetal group and the normal fetal group.The addition of two groups of exosomes to HUVEC showed that the growth restricted fetal umbilical cord blood exosomes inhibited the proliferation,migration and tubeforming ability of HUVEC.3.A total of 6 differentially expressed miRNAs were identified by miRNA sequencing analysis,all of which were up-regulated.GO showed high enrichment in phosphorylation,protein phosphorylation,nervous system development,protein binding and other aspects.KEGG showed high enrichment of pathways,calcium signaling pathways,and actin cytoskeleton regulation in cancer.Real-time PCR showed that miR-33b-5p was highly expressed in cord blood exosomes of growth-restricted fetuses.Overexpression of miR-33b-5p inhibited the proliferation,migration and tubeforming ability of HUVEC.4.Interaction between miR-33b-5p and IGF2BP1 was found through prediction and application of dual luciferase reporter gene detection.Real-time PCR showed that the expression level of IGF2BP1 in cord blood exosomes of growing-restricted fetuses was decreased.CCK-8,scratch test and angiogenesis test showed that miR-33b-5p had a negative regulatory effect on IGF2BP1,and further affected the protein expression of P-PI3 K and P-AKT in PI3K/AKT signaling pathway.Conclusion :Umbilical cord blood exosomes of fetuses with growth restriction can inhibit the proliferation,migration and angiogenesis of umbilical vein endothelial cells.This is related to the high expression of miR-33b-5p in umbilical cord blood exosomes.Further studies have shown that miR-33b-5p can negatively regulate IGF2BP1 and affect HUVEC proliferation,migration and angiogenesis through PI3K/AKT signaling pathway,thus leading to fetal growth restriction. |
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