| Objective:miRNA was extracted from amniotic fluid exosomes of Down syndrome(DS)fetus and normal fetus,and mi RNA sequencing technology was used to screen out differentially expressed miRNA,so as to investigate the role of miRNA in the growth and development of DS fetus.Methods:Collected women in the second trimester who were at high risk for noninvasive prenatal screening and required prenatal diagnosis.Amniotic fluid was extracted by amniocentesis under the guidance of B ultrasound.After centrifugation,supernatant was collected for exosome extraction,and cell precipitation was used for amniotic fluid cell culture.The cultured amniotic fluid cells were harvested with colchicine,prefixed,fixed,and G-banding,and the fetuses were divided into DS fetuses and normal fetuses.Amniotic fluid was collected from DS fetus and normal fetus in the second trimester of pregnancy,and exosomes of amniotic fluid were extracted by overspeed centrifugation.Set up control group: normal fetal amniotic fluid exosome;DS group: DS fetal amniotic fluid exosome.Amniotic exosomes were identified by transmission electron microscopy,nanoparticle tracking analysis(NTA)and Western blotting,respectively,and total RNA was extracted and purified.Differentially expressed mi RNAs in the control group and DS group were screened by mi RNA sequencing technology,and then target gene prediction,GO and pathway analysis of the differentially expressed mi RNAs were performed by bioinformatics methods.Among the differentially expressed mi RNAs,the three mi RNAs with the largest number of targeted DS key segments(mir-140-3p,let-7d-5p,mir-4512)were selected for RT-q PCR verification.The targeted regulation of let-7d-5p on BACH1 was verified by dual luciferase reporter gene technique.Results:Under transmission electron microscopy,exosomes in the control group and DS group were found to be between 30 nm-200 nm in diameter,with round or oval vesicles.NTA results showed that the peak size distribution of exosomes in control group and DS group was 143.1nm and 181.4nm,which was consistent with the distribution characteristics of exosomes diameter between 30-200 nm.Western blotting results showed that CD9,CD63,HSP70 and TSG101 were expressed in both the control and DS groups,and the expression levels were consistent.These results indicated that amniotic fluid exosomes were successfully isolated.mi RNA sequencing technology revealed that compared with the control group,there were 15 differentially expressed mirnas in exosomes in DS group,including 7 mirnas with up-regulated expression and 8 mirnas with down-regulated expression.Target gene prediction results showed that the differentially expressed mi RNA could target 17 DS related genes.GO analysis found that the main functions of target genes were related to the synapses of protein binding protein transport ATP binding transferase activity,and pathway analysis found that the enrichment of significant functional pathways was closely related to the development of nervous system.RT-q PCR results showed that mir-140-3p and let-7d-5p levels were significantly decreased in DS group compared with the control group(1.79 ± 0.21 vs 1.08 ± 0.03;1.42 ± 0.03 vs 0.19 ± 0.08,P< 0.05),consistent with sequencing results;The level of mir-4512 in DS group was significantly increased compared with control group(1.41 ± 0.15 vs 3.04 ± 0.05,P<0.05),contrary to sequencing results.Double luciferase reporter gene assay showed that let-7d-5p had almost no effect on empty vector.Compared with no-load group,let-7d-5p could regulate the expression of LUCiferase with 3’UTR in BACH1(P<0.001),fell by 62.08%;However,after the mutation of binding site,the regulatory relationship was weakened significantly(P<0.001),recovery 33.75%.Conclusions:Amniotic exosome let-7d-5p may be involved in the abnormal development of the brain nervous system in DS patients by regulating BACH1 expression,which has important significance for further elucidating the pathogenesis of DS. |
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