Abstract:

Objective To discuss the effect of exosomes （Exos） loaded with microRNA-520a-5p（miR-520a-5p） on the pregnancy outcomes in fetal mice with intrauterine growth restriction （FGR）， and to clarify its mechanism. Methods The mouse placental mesenchymal stem cells （MSCs） were cultured in vitro and transfected with miR-520a-5p adenovirus vector （Ad-miR-520a-5p） to obtain the Exos with high miR-520a-5p load （miR-520a-5p-MSCs-Exos）， which were then identified. The C57BL/6 mice were mated in cages at a female∶male ratio of 2∶1 to achieve successful pregnancy. Forty pregnant mice were divided into control group， FGR group， NC-MSCs-Exos group， and miR-520a-5p-MSCs-Exos group， with 10 mice in each group. Except for control group， the mice in other groups were exposed to excessive dexamethasone （DEX） during pregnancy to induce FGR models in the pregnant mice. The body weights of the fetal mice at birth and at 1， 2， 3， and 4 weeks after birth were detected； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of miR-520a-5p， DNA methyltransferase 3b（DNMT3b）， and vascular endothelial growth factor（VEGF） mRNA in placenta tissue of the mice in various groups； Western blotting method was used to detect the expression levels of DNMT3b and VEGF proteins in placenta tissue of the mice in various groups； methylation-specific PCR （MSP） was used to analyze the methylation rates of VEGF promoter in placenta tissue of the mice in various groups； dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-520a-5p and DNMT3b. Results The results of transmission electron microscope（TEM） and nanoparticle tracking analysis（NTA） showed that the Exos were spherical with particle size concentrated near 100 nm； the Western blotting method results showed that the surface biomarkers CD63 and CD81 of Exos were positively expressed. The RT-qPCR results showed that compared with NC-MSCs-Exos and Ad-NC-MSCs-Exos， the expression level of miR-520a-5p in Ad-miR-520a-5p-MSCs-Exos was increased （P<0.001）. The differences in birth body weight and the body weights at 1， 2， and 3 weeks after birth of the fetal mice among four groups were statistically significant （F=36.084， F=19.851， F=77.755， F=103.223； P<0.001）. Compared with control group， the birth body weight and the body weights at 1， 2， and 3 weeks after birth of the fetal mice in FGR group were decreased （P<0.05）； compared with FGR group and NC-MSCs-Exos group， the birth body weight and the body weights at 1， 2， and 3 weeks after birth of the fetal mice in miR-520a-5p-MSCs-Exos group were increased （P<0.05）. The One-way ANOVA results showed that the differences in the expression levels of miR-520a-5p， DNMT3b， and VEGF mRNA in placenta tissue of the mice among four groups were statistically significant （F=103.224， F=856.460， F=214.563； P<0.001）. The pairwise comparison between groups showed that compared with control group， the expression levels of miR-520a-5p and VEGF mRNA in placenta tissue of the mice in FGR group were decreased （P<0.05）， and the expression level of DNMT3b mRNA was increased （P<0.05）； compared with FGR group and NC-MSCs-Exos group， the expression levels of miR-520a-5p and VEGF mRNA in placenta tissue of the mice in miR-520a-5p-MSCs-Exos group were increased （P<0.05）， and the expression level of DNMT3b mRNA was decreased （P<0.05）. The One-way ANOVA results showed that the differences in the expression levels of DNMT3b and VEGF proteins in placenta tissue of the mice among four groups were statistically significant （F=245.601， F=149.360； P<0.001）. The pairwise comparison between groups showed that compared with control group， the expression level of DNMT3b protein in placenta tissue of the mice in FGR group was increased （P<0.05）， and the expression level of VEGF protein was decreased （P<0.05）； compared with FGR group and NC-MSCs-Exos group， the expression level of DNMT3b protein in placenta tissue of the mice in miR-520a-5p-MSCs-Exos group was decreased （P<0.05）， and the expression level of VEGF protein was increased （P<0.05）. The One-way ANOVA results showed that the difference in the methylation rate of VEGF promoter in placenta tissue of the mice among four groups was statistically significant （F=687.096， P<0.001）. The pairwise comparison between groups showed that compared with control group， the methylation rate of VEGF promoter in placenta tissue of the mice in FGR group was increased （P<0.05）； compared with FGR group and NC-MSCs-Exos group， the methylation rate of VEGF promoter in placenta tissue of the mice in miR-520a-5p-MSCs-Exos group was decreased （P<0.05）. Dual-luciferase reporter gene assay results showed that compared with miR-NC group， the luciferase activity in the cells containing DNMT3b-WT reporter vector in miR-520a-5p group was decreased （P<0.05）； compared with miR-NC group， the luciferase activity in the cells containing DNMT3b-MUT reporter vector in miR-520a-5p group had no change， no significant difference was observed （P>0.05）. Conclusion The MSCs-derived Exos highly loaded with miR-520a-5p may improve the pregnancy outcomes of FGR fetal mice by targeting and down-regulating the expression of DNMT3b， inhibiting VEGF methylation， and promoting VEGF expression.

Key words: Fetal growth restriction, Exosome, Mesenchymal stem cell, MicroRNA-520a-5p, DNA methyltransferase 3b, Vascular endothelial growth factor