| Decidualization is a complex and precise process,defects of decidualization may lead to pregnancy complications.Pre-implantation embryos are composed of inner cell mass and trophoblast cells.The paracrine signals produced by trophoblast cells regulate the decidualization,and the inner cell mass can regulate the proliferation and differentiation of trophoblast cells.However,there were few studies on the role of inner cell mass in decidualization.Tetraploid embryos,which inner cell mass was defective,have functional trophoblastic cells.Decidua induced by diploid embryos and tetraploid embryos were compared to investigate whether the inner cell mass had a regulatory role in the decidualization.Mice were employed to investigate the role of inner cell mass in decidualization.Decidua were induced by tetraploid embryos?inner cell mass defective embryos?and diploid embryos?normal 2-cell embryos?,respectively.The decidualization was compared from embryo implantation,decidual distribution and decidual morphology at 4.5 and 7.5 of pregnancy.Small RNA sequencing technology was used to analyze the differentially expressed miRNAs between the two groups,target genes of differentially expressed miRNAs were predicted,the main functions of target genes and their enriched pathways were analyzed.Sequencing results were verified by real-time fluorescence quantitative,the dual-luciferase activity reporting experiment and real-time fluorescence quantitative were used to verify the targeted regulatory relationship between miRNA and target genes.The role of miRNAs in decidualization was explored by the injection of miRN A antagomir.The results showed that diploid embryo-induced decidua and tetraploid embryo-induced decidua were similar in embryo implantation site and decidual distribution.They were evenly distributed throughout the uterus,there were cracks on the membrane surface in the tetraploid embryo-induced decidua.It was found that the division of tetraploid embryo-induced decidua were more vigorous than diploid embryo-induced decidua by paraffin sections.Observation of the internal structure of decidua revealed that there was no complete fetal structure inside the decidua induced by tetraploid embryos.In the internal structure of decidual tissue induced by diploid embryos,while the complete embryonic structure could be seen in the decidua induced by diploid embryos.Through small RNA sequencing technology,we found that there were 16 differentially expressed miRNAs between the two groups,there were 11 miRNAs up-regulated in decidua induced by tetraploid embryos,including miR-878-5p,miR-433-5p,miR-741-3p,miR-302a-5p,miR-466i-5p,miR-302d-3p,miR-466f-3p,miR-465c-5p,miR-7068-3p,miR-302a-3p and miR-144-5p.There were 5 miRNAs down-regulated in the tetraploid embryo-induced decidua,including miR-690,miR-363-3p,miR-147-3p,miR-193b-5p and novel327.Target genes of differentially expressed miRNAs were mainly involved in protein binding function and enriched in estrogen and VEGF signaling pathway.miR-690 was the most differentially expressed miRNA between the two groups,31 target genes of miR-690 were predicted.RelA was selected as a candidate target gene for subsequent study.We found that RelA and miR-690 had opposite gene expression patterns by qRT-PCR.It was found that the relative luciferase activity of the recombinant plasmid of wild-type RelA3 'UTR transfected with the mir-690 mimic was significantly lower than that of the negative control group?P<0.05?,there were not significant difference in other groups?P>0.05?.These results indicated that miR-690 can bind to the position of RelA 3'UTR 326-333?UAGCCUU?to target negative regulation of RelA.The number of decidua in mice injected with miR-690 antagomir was significantly lower?P<0.05?than the control group,pregnancy failure appears with increasing concentration of antagomir.In summary,the defect of the inner cell mass causes an abnormal decidualization,mir-690 can negatively regulate the expression of RelA and play an important role during decidualization. |
PDF Full Text Request