Expression And Role Of Mmu-miR-141in Endometrium Of Early Pregnant Mouse During Embryo Implantation

The successful implantation of embryos is depended on the synchronizedreciprocal interaction between blastocyst and endometrium. During thisprocess, differential expression of a series of cytokines which are related tophysiological changes of the endometrium play a key role,while morestudies demonstrate that microRNAs (miRNAs) play an important role inthe fine regulation of differential expression of genes. Our project teampreviously study expression of miRNAs in endometrium of early pregnantmouse before and after the embryo implantation with miRNAs chiptechnology and show that the mmu-miR-141expression in endometrialtissue after the implantation (d6) was obviously lower than before theimplantation(d4). In this article, mmu-miR-141was designed to detect theexpression and role of mmu-miR-141in endometrium of early pregnantmouse during embryo implantation,which will provide the experimentalbasis for further understanding of the molecular mechanism of embryoimplantation. Methods1. Fluorescence quantitative polymerase chain reaction (FQ-PCR)was choosed to verify the microarray results，and in situ hybridization wereapplied to detected the location of mmu-miR-141in endometrium of earlypregnant mouse before and after the embryo implantation.2. MTT assay was applied to detect effect of mmu-miR-141inhibitor onendometrial Stromal Cells’cycle and flow cytometry was choosed to detectthe effect of mmu-miR-141inhibitor on endometrial Stromal Cells’cycle andapoptosis.3. The application of target gene prediction database of miRNAs tosearch target genes of mmi-miR-141.4. Mimics and inhibitor of mmu-miR-141were transiently transfectedinto endometrial stromal cells，and then Western blot was applied to detectthe expression levels of PTEN.5. In vitro，mmu-miR-141inhibitor or its mimics was injected into theuterine horns of mice on pregnant d2, mice were killed on pregnant d7andthen count the number of embryos.Results1. FQ-PCR results show that the expression of mmu-miR-141inendometrial tissue in pregnant d6group was lower than in pregnant d4group(P <0.05), which consistents with the microarray results.2. In situ hybridization results suggested that the mmu-miR-141wasmainly located in the stromal cells, almost no expression in the luminal epithelium and glandular epithelium. expression intensity of pregnant d6group was decreased compared with pregnant d4group (P <0.05).3. mmu-miR-141inhibitor was transiently transfected into endometrialstromal cells, MTT assay results showed that reduced mmu-miR-141coulddecrease proliferation activity of endometrial stromal cell (P <0.05); flowcytometry results showed that when mmu-miR-141was downregulated, thecells were arrested in S period, and its apoptosis rate increasedsignificantly(P <0.01).4. Bioinformatics analysis and preliminary experimental resultssuggested that PTEN， PDCD4and KLF are the regulated targets ofmmu-miR-141.5. mimics or inhibitor of mmu-miR-141was respectively transfectedinto endometrial stromal cells, we found that raised and loweredmmu-miR-141does not change the level of PTEN mRNAexpression.Meanwhile, downregulated mmu-miR-141resulted in theexpression of PTEN increased significantly, while upwnregulatedmmu-miR-141had a little effect on the expression level of PTEN.6. Both of mmu-miR-141inhibitor and its mimics can result inreduction of number of embryo implantation.Conclusion1. Significant differences of mmu-miR-141expression level exist inendometrial tissue of early pregnant mice before and after the embryo implantation, mmu-miR-141expression level was lower afterimplantation(d6) than that before implantation(d4).2. mmu-miR-141regulate proliferation and apoptosis of endometrialstromal cells by targeting PTEN，and then affect embryo implantation.