The Functional Characterization Of MiR-183-5p In Mouse Embryo Implantation

MicroRNA(miRNA)is a kind of noncoding single stranded RNA with 18-24 nucleotides encoded by endogenous gene.It inhibits translation through specific complementary binding with the 3’-UTR of the target gene,and finally regulating the expression of the target gene at the post transcriptional level.Studies have shown that miRNA plays an important role in post transcriptional regulation during embryo implantation.However,miRNAs which may play regulatory role during the window of implantation remains to be further explored.ICR strain mice were used to research the miRNA expression in different days of early pregnancy in mice,and the uterine tissues of mice on D1,D4 and D5 were analyzed by small RNA sequencing analysis.The uterus tissue on D5 was divided into D5IMS and D5IIS.Functional annotation and pathway analysis of the differentially expressed miRNAs were carried out,and the effect and molecular mechanism of miR-183-5p on embryo implantation were further revealed.We explored the miR-183-5p expression changes in different uterine state of early pregnancy.Through the Transwell migration and invasion experiment,scratch healing experiment and cell apoptosis experiment,we explored the biological function of miR-183-5p in the process of embryo implantation.Through the prediction of the target genes of miR-183-5p,the target relationship between miR-183-5p and the predicted target genes were verified by the double Luciferase Report assay.The main results are as follows:(1)Ten miRNAs with different expression were screened out from different days of early pregnancy.Eight of them were down-regulated(miR-182-5p,miR-183-5p,miR-375-3p,miR-192-5p,miR-429-3p,miR-210-3p,miR-31-5p,miR-200a-5p),and two were up-regulated(miR-199b-5p,miR-218-5p).The change trend of miR-183 family(miR-183-5p,miR-96-5p and miR-182-5p)in early pregnancy of mice was consistent.Compared with pregnancy D1,the expression of miR-183 family in mouse uterus decreased gradually(P<0.001)in D1-D4.The expression level of miR-183-5p family in implantation site was lower than that in interimplantation site,but the difference was not significant.(2)The change of miR-183-5p in early pregnancy was mainly due to the regulation of the maternal uterus itself.The expression of miR-183-5p could be further reduced by the presence of active embryos.The results of hormone model showed that P4 could reduce the expression of miR-183-5p(P<0.05).In the early stage of embryo implantation(D1-D4),the expression of miR-183-5p in the uterine cavity epithelium and glandular epithelium gradually disappeared,and the expression of miR-183-5p in the basal stromal cells gradually increased.After implantation(D5),miR-183-5p was mainly expressed in the superficial stromal cells at the implantation site and in the glandular epithelial cells and stromal cells at the interimplantation site.After increasing the expression level of miR-183-5p in uterus,the embryo implantation site disappeared,and the receptivity of mouse uterus to embryo implantation decreased.Overexpression of miR-183-5p can reduce cell migration/invasion ability and cell activity,thus inhibit embryo implantation.(3)Hbegf,Lamc1,Zeb1,Zeb2 and Arid5b were the predicted target genes of miR-183-5p.The changes of mRNA expression of the five predicted target genes in the early pregnancy of mice were contrary to that of miR-183-5p.Hbegf and Lamc1 are the downstream target genes of miR-183-5p.Overexpression of miR-183-5p can increase the expression of Zebl and Zeb2.To sum up,miR-183-5p can inhibit embryo implantation during the process of embryo implantation.In the early stage of pregnancy,the expression level of miR-183-5p in mouse uterus will gradually decrease under the conditions of maternal regulation,embryo,hormone and reach a low level before implantation,so as to promote embryo implantation.Hbegf and Lamcl are the downstream target genes.MiR-183-5p inhibits embryo implantation by reducing the recognition and adhesion between uterus and embryo.This study revealed the changes of miRNA in mouse uterus during early pregnancy,and preliminarily elucidated the effect and mechanism of miR-183-5p in embryo implantation,providing a new target and research strategy for improving the efficiency of embryo implantation.