The Function Of MMU-MIR-193 In Mouse Uterus During Embryo Implantation

Objective:Application of Real-time RT-PCR and in situ hybridization to confirm the expression rule of mmu-miR-193 in the before and after embryo implantation (pregnancy D4, D6) mouse endometrial which was screening by miRNAs microarray was the most differentially expression. We predicted its possible target genes by database search. In vitro by transfection of mmu-miR-193 mimics and inhibitors, verify whether the Grb7 regulated by mmu-miR -193.pregnant Second day in the uterine horn by injection of mouse mmu-miR-193 agomir to study the effect on embryo implantation. To clarify the mechanism of normal human reproductive process and diagnosis and treatment of diseases reproductive provided the basis.Methods:1. Specimen collection: Established NIH mouse model of pregnancy, separation endometrial tissue of pregnant d4, d6 days. and then immediately frozen at -80℃for further test.2. Real-time RT PCR detected the differentially expressed of mmu-miR-193: according to the mmu-miR-193mature sequence to design specific reverse transcription primers, design its upstream and downstream primers for real-time RT PCR.3. In situ hybridization detected mmu-miR-193 in the before and after embryo implantation (pregnancy d4, d6) endometrial expression site.4. Bioinformatics analysis: Application miRNAs target gene prediction database website Pictar, miRGen, Targetscan to retrieve mmu-miR-193 predicted target genes.5. Transfection test: The transfection of mmu-miR-193 mimics and inhibitors enhance or reduce its function, Western blot after transfection was detected the changes in protein expression of GRB76. Function test: Day 2 of pregnancy in the mouse uterine horn by injection of mmu-miR-193 agomir, Day 7 of pregnancy Caesarean section counts the number of embryo implantation.Results:1. Real-time RT-PCR result was consistent with the microarray results; mmu-miR-193 was significantly increased in D6 endometria compared with D4.2. In situ hybridization showed mmu-miR-193 in mouse endometrial stromal cells, luminal epithelium and glandular epithelial cells were expressed.3. Use bioinformatics, predicted by the database, get Grb7 as the mmu-miR-193 target genes, suggesting mmu-miR-193 by regulating these target genes to regulate embryo implantation.4. In vitro, we adopted transfection mmu-miR-193 mimics and inhibitors, confirmed that Grb7 is mmu-miR-193 regulated target genes.5. Day 2 of pregnancy in mice, In the uterine horn by injection of mmu-miR-193 agonist, leading to a decline in the number of embryo implantation.Conclusion:1. mmu-miR-193 was significantly different expression in the before and after embryo implantation endometrial. After implantation of the embryo were significantly more elevated pre-implantation.2. mmu-miR-193 by regulating the expression of its target proteins GRB7 thereby regulating embryo implantation.