Mouse Endometrium Spatiotemporal Expression MRNA And MicroRNA Associated With Embryo Implantation And SPOP Influnce Endometrium Stromal Decidualization

Part I MOUSE ENDOMETRIUM TEMPORAL AND SPATIAL EXPRESSION MRNA AND MICRORNA ASSOCIATED WITH EMBRYO IMPLANTATIONObjective:Embryo implantation is a dynamic physiological process involving morphological and molecular changes in the endometrium during the pre-receptivity, receptivity and implantation phases.A comprehensive analysis of mRNA and microRNA (miRNA) profiles during implantation will likely provide new clues to elucidate the underlying mechanisms governing embryo implantation.Methods:Collect the endometrial tissues of the pregnancy dl, pregnancy d4 and the implantation site (IS) and inter-implantation site (IIS) of the pregnancy d5. Extraction the total RNA of endometrial tissues of the dl, d4 and the implantation site (IS) and inter-implantation site (IIS) of the endometrium on d5. Next Generation Sequencing was employed to test the profile of mRNAs and miRNAs of d1, d4, d5IS and d5IIS of the endometrium on d5. RT-PCR Verification:screen the negatively related miRNA-mRNA pairs from the most likely targets of differentially expressed miRNAs based on the expression values of the miRNAs and mRNAs from NGS. Random selection 5 couple of miRNA-mRNA. Real-time quantitative PCR was performed on selected miRNAs and their predicted target mRNAs to validate their negatively correlated expression. The results of NGS be confirmed reliabilit miRNA and mRNA has a potential relationship in embryo implantation. The profile of mRNAs of d1、d4、d5IS and d5IIS were compared to screen out the differential gene expression. The Venn diagram were recruit to dynamic analysis the gene of peri-implantation. The profile of miRNAs of dl、d4、d5IS and d5IIS were compared to screen out the differential miRNAs expression.,To recruit the tool of Targetscan database、miRanda database and DIANA-microT 3.0 to predict the target gene of miRNA. Intergrate analysis the profile of miRNAs and mRNAs and screen out the negatively related miRNA-mRNA pairs.Result:The NGS indicate that the miRNAs expression of d1、d4、 d5IIS�'�d5IS have significant difference. The Endometrium of d1 and d5IS express up-regulated miRNAs, while the Endometrium of d4 express down-regulated miRNAs, which suggestion miRNAs play different role during peri-implantation.5720 target gene were predicted in 63 significant difference expression miRNAs. Intergrate analysis the profile of miRNAs and mRNAs and screen out 1473 pairs of negatively related miRNA-mRNA pairs.among 1473 pairs of miRNA-mRNA,138 pairs of miRNA-mRNA always present negative correlation. Random selection 5 couple of miRNA-mRNA Real-time quantitative PCR was performed on selected miRNAs and their predicted target mRNAs to validate their negatively correlated expression and To confirm the results of NGS are reliability. The miRNA-mRNA interaction network unraveled the regulatory relationship between miRNAs and mRNAs. Hub miRNAs (mmu-miR-96 and mmu-miR-200b) were identified to target Bcl-2, Klf13 and PGR, which are associated with the preparation of the receptive condition or the maintenance of early pregnancy.Conclusion:The NGS indicate that the mRNAs Temporal and Spatial expression of d1、d4、d5IIS�'�d5IS have significant difference, which result in the endometrium play different role during the peri-implantation. Since the gene of Bcl-2, Klf13 and PGR which target the hub miRNA have associate with the state of receptivity during implantation. It is indicate that those gene paly important role in optimize receptivity and Maintain the early pregnancy MiRNAs always regulted the mRNAs expression during whole implantation.Part Ⅱ SPOP INFLUNCE ENDOMETRIUM STROMAL DECIDUALIZATIONObjective:For successful embryo implantation the decidualization of endometrium stromal is a prerequsite event, which involve proliferation and Differentiation of endometrium stromal induce by steroid hormones. Previous studies indicated the significant differential expression of SPOP in embryo implantation. SPOP associated with ubiquitination, while the relation between SPOP and Implantation haven’t report. So the aim of our study is to to clarify the relationship between SPOP and implantation.Methods:Mature female mice mated with vasectomy male mice to establish the model of psedudopregnant mice. Arificially induced decidualization was obtain by intrauterine injected corn oil.In order to establish the artificial hormone regulation model estradiol and progesterone were subcutaneous injected in female mice after removal the ovaries. The RT-PCR, Western blot and immunohistochemistry technique was used to detect the expression pattern of SPOP mRNA and protein in endometrium of peri-implantation mice, pseudopregnant mice and steroid hormones regulation of mice. Inorder to detected the expression of SPOP in the artificial decidualization cells siRNA technology were perform. Detected the expression decidualization marker in decidual cell wich was knockout SPOP by siRNA. Flow cytometry detected the apoptosis of stromal cellResult:During the peri-implantation period SPOP with fluctuated expression which with low expression level in dl, d2 and d3 and high expression level in d4,d5,d6, then with fall expression in d7 while pseudopregnant mice have the same expression pattern. After artificial decidualization the mice endometrium express high level of SPOP. The SPOP expression was regulted by estrogen and progesterone. The decidual markers were significantly reduced in decidual cells after knockout SPOP. SPOP inhibited apoptosis of stromal cell.Conclusion:Both Estrogen and Progesterone could regulate the expression of SPOP. SPOP could promote endometrium stromal cell the different into decidual cells by effect of apoptosis and ubiquitination.