Preliminary Study Of Estrogen And Progesterone On Micrornas Regulation And Effect Of Mmu-microrna-200a Overexpression On Mouse Embryo Implantation

PART IPRELIMINARY STUDY OF ESTROGEN ANDPROGESTERONE ON MICRORNAS REGULATIONBackground: Successful pregnancy is dependent on a healthy uterus thatis fit to receive and support a fertilized embryo. The uterus is an endocrineorgan, responsive to the presence of the ovarian steroid hormones, estrogenand progesterone, which activate transcription of target genes through thebinding of their cognate receptors, the estrogen receptor and theprogesterone receptor. Estrogen and progesterone signaling have beendemonstrated to be critical for the initiation and maintainance of pregnancy.MicroRNAs(miRNAs) regulate the expression of many genes, and previousstudies have demonstrated the relationship between miRNAs expressionand embryo implantation. However, there is little report about whoregulates miRNAs expression. Because many crucial genes which areinvolved with implantation are regulated by E2and P4. Then, the purpose of this study is to explore whether E2and P4affect implantation throughregulating miRNAs expression.Methods: Collected mouse uterus endometria tissues on pregnancyD1and D6to mimic estradiol and progesterone regulate model (E2levelreaches a peak on D1, and P4is very low; instead, P4level reaches a peakon D6, and E2is very low). Then extracted total RNA to performmicroRNA sequencing. At last, the results were analyzed by Bioinformatics.Results: The results of miRNA sequencing showed that6miRNAexpression was up-regulated more than2fold and73miRNA expressionwas down-regulated more than2fold when D6versus D1, and screen forthe target genes of these differential miRNAs are involved in whichsignaling pathways. The target genes of these differential miRNAs areinvolved in metabolism, MAPK, tight junction signaling throughBioinformatics analysis.Conclusions: These results preliminarily reflect in mouseendometrium, the expression of miRNAs is regulated by estrogen andprogesterone. Some miRNAs are differentially expression in endometriumon pregnancy D1, and D6and the target genes of these miRNAs areinvolved in several signaling pathways which are very important during theimplantation process through Bioinformatics analysis. This suggested thatmiRNAs have a role in mouse implantation. PART IIEFFECT OF MMU-MICRORNA-200A OEREXPRESSION ONMOUSE EMBRYO IMPLANTATIONBackground: Successful mouse embryo implantation requires areceptive uterus and an activated blastocyst. A large number of genes,cytokines and other factors are involved with the process.MicroRNAs(miRNAs) regulate the expression of many genes, and previousstudies have demonstrated that miRNAs regulate embryo implantationthrough their target genes. However, there is much less report about specificmiRNA involved in this process. According to previous results, we foundthat mmu-miR-200a expression was down-regulated by P4and expresseddifferentially before and after implantation. Moreover, studies have alsoshown that miR-200a was differentially expression in many endometrialdeseases (such as endometrial carcinoma and endometriosis). Thus, thisstudy is to explore the role of mmu-miR-200a during embryo implantationand its target gene, and clarifying the physiological functions of uterinemiRNAs will elucidate the embryo implantation process, and may evencontribute to curing infertility and inventing new contraceptives.Methods: Real Time-PCR examined mmu-miR-200a expression levelon pregnancy D4, D5and D6, and in the implantation sites andinter-implantation sites in the uterine endometria on pregnancy D5. Then we performed in situ hybridization to detect the expression pattern ofmmu-miR-200a. The target gene of mmu-miR-200a was verified bybioinformatics analysis, primary stromal cell culture, transfection andfluorescence intensiy assay. The implantation rate was examined byinjecting lentivirus which was designed to promote overexpression ofmmu-miR-200a in the area of injection. Finally, we examined the apoptosisrate of primary stromal cells after tranfection with mmu-miR-200a control,mimic or inhibitor through flow cytometry.Results: Here it showed that the expression of mmu-miR-200a wasmuch lower at implantation sites than that at inter-implantation sites onpregnancy D5. Mmu-miR-200a had the highest level of expression on D4,and the expression levels decreased gradually on D5and D6, and it wasmainly expressed in stromal cells and weakly expressed in epithelial andglandular cells. PTEN, as the target gene of mmu-miR-200a, had the sameexpression location and the opposite expression level with mmu-miR-200a.Moreover, the implantation rate was decreased by injecting mmu-miR-200aoverexpression lentivirus. Additonally, when transfection withmmu-miR-200a inhibitor, the apoptosis rate of stromal cells was increaseobviously and PTEN protein expression level was up-regulated. Instead,When transfection with mmu-miR-200a mimic, the apoptosis rate ofstromal cells was decrease and PTEN protein expression level wasdown-regulated. Conclusions: This study demonstrated that overexpression ofmmu-miR200a leads to decreased implantation rate. According to theexpression pattern of mmu-miR-200a and its target gene PTEN, and therate of apoptosis of primary stromal cells by transfection withmmu-miR-200a mimic and inhibitor, we believed that mmu-miR-200aregulate moderate proliferation of stromal cells to ensure normal embryoimplantation process through PTEN.