The Different Expression Of MICRORNAS And MIR-106B Expression Rule Study In Endometrium Of Early Pregnant Mouse During Embryo Implantation

Objective:To explore the rule of miRNAs in endometria of early pregnant mouse, miRNAs microarray was used to investigate the differentially expressed miRNAs in the mouse endometria from pregnant D4 and D6, real-time fluorescence quantitative PCR (RT-FQPCR)was used to confirm the microarray, and we predicted the possible target genes by combination anticipated algorithms,in order to study the role of miRNA in implantation of early pregnant mouse; through the miRNAs target gene database to find the target genes and detect the expression of miR-106b ,which taget genes were associated wiht proliferation of endometria, in the mouse ednometria from pregnant D4 and D6, in order to study the mechanism to the mouse endometria of miR-106b in implantation of early pregnant mouse.Methods:1. Sample preparation: collect the mouse endometria from pregnant D4 and D6. 2. MiRNAs microarray analysis: Extraction the total RNA of mouse endometrial tissue, use Cy3 from miRCURYTM LNA Array labeling kit (Exiqon, Denmark) label the total RNA according to the instructions. Cy3 labeled RNA use miRCURYTM LNA Arrays (Exiqon, Denmark) perform hybridization. By Gene Pix 4000B scanner and the Gene Pix 6.0 software (Axon Instruments, Union City, CA) for image scanning and data analysis.3. Fluorescence quantitative PCR detect the differentially expressed miRNAs: according to the mature miRNA sequence to design specific reverse transcription primers for reverse transcription, design miRNA upstream and downstream PCR primers for RT-FQPCR, using U6 RNA as the endogenous reference for normalization.4. Bioinformatics analysis: use the most commonly used miRNAs target gene prediction website Targetscan, Pictar, miRBase to search the predicted target genes of differentially expressed miRNAs.5. In situ hybridization: in situ hybridization detect the expression of miR-106b in the mouse endometria from pregnant D4 and D6.Results:1. The miRNAs microarrays results showed that there are 17 miRNAs up-regulated 2 times and above in D6 endometria, and 18 miRNAs down-regulated 2 times and above ,compared with D4 endometria. The RT-FQPCR results confirmed the microarray results. Through Targetscan, Pictar, miRBase database, we found some target genes of differentially expressed miRNAs, which are associated with proliferation,apoptosis and anti-apoptosis.2. MiRNAs microarray , RT-FQPCR and in situ hybridization results showed that the expression of miR-106b in D4 endometria is 2 times above than in D6 endometria and the miR-106b were mainly expressed in endometrial stromal cells, the luminal epithelium and glandular epithelial cells do not express. It hints that miR-106b may be through regulate its target genes to regulate early pregnancy endometrial stromal cells growth, proliferation, differentiation and apoptosis.Conclusion:1. This study showed that compared with D6 and D4, there were differential expression of miRNAs, miRNAs may be play an important role in endometria during embryo implantation.2. Using RT-FQPCR to further verify the differential expression miRNAs, and dectecd the expression of these miRNAs in the mouse endometria from pregnant D4 and D6.3. Use bioinformatics methods to predict target genes of differentially expressed miRNAs, presume that these miRNAs through regulate its target genes associated with embryo implantation to regulate embryo implantation.4. There were differentially expression of miR-106b in the mouse endometria from pregnant D4 and D6 by RT-FQPCR, suggesting that it may be regulate relevant target genes and then regulate the embryo implantation. In situ hybridization detection of miR-106b were expressed mainly in endometrial stromal cells in mice, according to the database and some literatures analysis suggesting that miR-106b could be regulate the expression of target genes Npm1, Tsg101 and PTEN to regulate endometrium stromal cell growth, proliferation, differentiation and apoptosis and then regulate embryo implantation.