The Differential Expression Of Micrornas Between Implantation Sites And Inter-implantation Sites In Early Pregnancy In Mice And Their Potential Functions

ObjectiveA series of changes occur in the endometrium to accept the embryo duringthe window of implantation, and successful implantation of the embryo dependson the different gene expressions between implantation sites andinter-implantation sites. As modulators of gene expression, microRNAs(miRNAs) have been identified expressed differently and play a regulate roleduring embryo implantation. To better understand how miRNAs regulateimplantation and the related molecular mechanisms, microarrays were performedto compare the expression profiles of miRNAs and mRNAs between implantationsites and inter-implantation sites in the endometrium of pregnant mice on Day5.Differently expressed pathways at implantation sites were got by combining theresults of the microarray experiments, and key miRNAs that may play animportant role were found. It would lay the foundation for studying molecularmechanisms of embryo implantation in mice.Methods1. Sample collection: Collect the endometrial tissues of implantation sites and inter-implantation sites of pregnant mice on Day5.2. microRNAmicroarray analysis： Extraction the total RNAof tissuesby Trizol reagent, Label the total RNA with miRCURYTM Array PowerLabeling kit (Cat#208032-A, Exiqon, Denmark) according to the protocol,miRCURYTM Array (Exiqon, Denmark) was used to performhybridization with the labeled RNA. Image scanning was carried out byAxon GenePix4000B microarray scanner and GenePix pro V6.0softwarewas for data analysis.3. Gene microarray analysis: The total RNA was extracted by Trizolreagent, and then was labeled with Quick Amp Labeling Kit(Agilent),Agilent Gene Expression Hybridization Kit (Agilent) was used tohybridized with the labeled RNA. After washed by Gene Expression WashBuffer(Agilent), image scanning was performed by microarray scanner. Atlast, the data was analyzed by Agilent Feature Extraction.4. Confirmation of Real-time PCR: Designed the specific reverseprimers and a pair of PCR primers according to the mature miRNAsequence, U6was used as control to perform real-time PCR. Genes wereselected randomly to perform real-time PCR.5. Bioinformatics analysis: The target genes of differently expressedmiRNAs were got by predicted using taegetgene prediction websiteTargetscan, Pictar and miRBase, and then the gotten targets were comparedwith the results of gene chips. Potential target genes of miRNAs in endometrium of Day5were obtained by getting the intersection ofpredicted targets and differentially expressed genes. Database GO andKEGG were used to analyze the function of the obtained target genes, andgot the key miRNAs by drawing the negative regulatory network usingnegative correlation operation.Result1. The results of miRNA microarray showed that there were30up-regulated miRNAs and42down-regulated miRNAs at implantation sitescompared with inter-implantation sites.2. The real-time PCR results showed that the expression trends ofmiRNAs and mRNAs selected randomly were consistent with microarraydata.3. Bioinformatics analysis: Potential target genes of miRNAs inendometrium of Day5were obtained by getting the intersection of predictedtargets and differentially expressed genes. The functional analysis showedthat there were20up-regulated pathways and14down-regulated pathwaysat implantation sites. The key miRNAs were got by drawing the negativeregulatory network using negative correlation operation.Conclusion1. The results of this study confirmed that there were differentlyexpressed miRNAs between implantation sites and inter-implantation sitesin the endometrium of pregnant mice on Day5(including up-regulated miR-21、miR-96、miR-30b and down-regulated miR-298), that suggestmiRNA may play a role during embryo implantation.2. Real-time PCR was used to detect the expressions of some differentlyexpressed miRNAs and genes, the results confirmed that the data ofmicroarrays was reliable.3. The negative related target genes of differently expressed miRNAswere obtained by bioinformatics analysis. In addition, we got theup-regulated pathways at implantation sites (cell adhesion molecules andnatural killer cell mediated cytotoxicity) and the key miRNAs (miR-96、miR-30b) that may play an important role. miRNAs may modulate embryoimplantation by related signaling pathways.