The Identification And Functional Analysis Of Long Non-coding RNAs In Mouse Uterus During Implatation Window

Successful implantation is dependent on the intricate molecular dialogue between the receptive uterus and active blastocyst.The establishment of a receptive uterus is an essential step for successful embryo implantation in mammalian reproduction,which only occurs in a spatiotemporally restricted time known as the implantation window.Although fertilization in vitro and embryo transfer technology have been widely used in clinical practice,the pregnant rate is still unsatisfactory.Poorly uterine receptivity is considered as one of the major cause for the failure of assisted reproductive techniques.Previous research showed protein encoding genes and miRNAs in endometrial receptivity play an important role in embryo implantation and subsequent decidualization.Long non-coding RNAs?lncRNAs?is as the largest numbers of non coding RNA family.The function of lncRNAs in oocyte maturation and early embryo development has been studied,but the role of lncRNAs during embryo implantation has rarely been reported.In this study,the expression profiles of lncRNAs in uterine samples from pregnant mice on day 4?D4U?,day 5 at implantation sites?D5IU?and inter-implantation sites?D5NU?were conducted via Illumina high-sequencing technique,which lays the foundation for the study of mechanism of lncRNAs in endometrial receptivity and subsequent decidualization.The main results obtained by this experiment as follows:1 The map of lncRNAs in mouse uterus during the implantation window was built up by Illumina HiSeq^TM 2500 high-throughput sequencing platform,and a total of 7,764 putative lncRNA transcripts were identified,including 6,179 known transcripts and 1,585 novel transcripts.13 selected lncRNAs were detected using qRT-PCR and the validation results showed that the expression patterns of these lncRNAs were in excellent agreement with the RNA-seq data.2 The expression level,sequence and conservation of lncRNAs were further analyzed.The target genes of lncRNAs in cis or trans were predicted.We further comprehensively identified and characterized the biological functions of 74 differentially expressed lncRNAs by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis,and the results showed that the differentially expressed lncRNAs were mainly involved in the tissue remodeling,immune response and metabolism related processes.Meanwhile,the differentially expressed lncRNAs regulated multiple signaling pathways and might play an important role in the regulation of embryo implantation.3 The known lncRNA GAS5 in our study was significantly upregulated in D4 U,and differentially expressed between D5 IU and D5 NU according to RNA-seq results.Furthermore,the dynamic expression pattern of GAS5 in the uterus during the implantation window was revealed using early pregnancy,delayed implantation and activation and steroid treatment models.The expression of GAS5 in the pregnant uterus was regulated by the active embryo and progesterone.The above results indicated that GAS5 may play a role in the establishment of endometrial receptivity and embryo implantation process.4 The result of FISH revealed that GAS5 mainly expressed in the nucleus of mouse endometrial stromal cells.The expression level of GAS5 in the uterus of artificially induced decidua was significantly up-regulated,and observed significant decrease in a time-dependant manner during the process of decidualization in vitro,which indicated that GAS5 may play a role durting decidualization.In conclusion,our study established the lncRNAs library and screened out differently expressed lncRNAs during mouse implantation window.Functional annotation and pathway analysis of differentially expressed lncRNAs were done.GAS5 may be involved in the regulation of embryo implantation and decidualization in mice,and GAS5 may be regulated by active embryo and progesterone in early pregnancy process.