Research On The Regulatory Mechanism Of Differentially Expressed MiRNA And LncRNA In The Receptive Endometrium Of Dairy Goats

During embryo implantation,impaired endometrial receptivity is one of the major reasons for the failure of embryo implantation,which would suffer economically in livestock production.Accumulated evidence suggests that the establishment of endometrial receptivity is a complicated dynamic process involving multiple factors.In recent years,miRNA were found to play an important role in endometrium development,but only few studies were performed on ruminant,and the regulation mechanism in dairy goat is still unclear.Long noncoding RNA(lncRNA)can act as miRNA molecular sponge to regulate miRNA function,but at present,whether there are lncRNA existing in dairy goat endometrium or not,and the function of lncRNA remains unknown.In this study,the expression of mi RNA and lncRNA in pre-receptive(PE,D5)and receptive(RE,D15)endometrium of dairy goat were analyzed by high-throughput sequencing.The function mechanism and the relationship between lncRNA and miRNA on endometrial cells were detected by methods of overexpression or interference,double luciferase gene reporter system,MTT,Flow cytometry,RT-qPCR,and Western blot.Regulation networks of lncRNA-miRNA-mRNA were constructed for the mechanism of endometrial receptivity in dairy goat.Thus,elucidating the physiological roles of endometrial mi RNA-lncRNA would help us better understand the molecular mechanism of endometrial receptivity.Our main results are as follows:1.The endometrium reached the receptive state at gestational day 15(D15)in dairy goat.In dairy goat,the endometrial thickness was significantly increased at D15 compared to D5,and well-formed pinopodes were found on the surface of endometrium at D15 with higher E2 and P4 concentrations in the serum and ERα(Estrogen Receptor alpha),ERβ(Estrogen Receptor beta)in the endometrium.Moreover,some expression levels of marker genes of receptive endometrium(OPN,VEGF and PRL)increased at D15 compared to D5,and significant differences were observed in the cell proliferation and apoptosis in the uterus.Based on the results of this study it can be safely concluded that the endometrium developed into the receptive state at gestational D15 in dairy goat.2.Analysis of the differentially expressed miRNA in pre-receptive(PE,D5)and receptive(RE,D15)endometrium of dairy goat.High-quality miRNA were detected in receptive and pre-receptive goat endometrium,and we identified 545(50.98%)known miRNA in the RE and 522(61.63%)in the PE.There were 145 differentially expressed miRNA in RE compared with PE meeting the criteria of P-value < 0.05.And more notably,miR-26 a level significantly decreased in the RE compared with PE(P-value = 0.00).Moreover,GO and KEGG analysis of the target genes of the differentially expressed miRNA revealed some candidate miRNA,genes and pathways that may involve in the formation of endometrial receptivity.3.miR-26 a regulated the endometrial cells via the PTEN-PI3K/AKT pathway.In this study,miR-26 a was widely expressed in dairy goat,and was found to be regulated by β-estradiol(E2)and progesterone(P4)in endometrial epithelium cell(EEC)as well as stromal cell(ESC).And the decrease of miR-26 a in RE(D15)was mainly due to the decreased in ESC.Furthermore,miR-26 a played a role in the regulation of cells proliferation and apoptosis by directly regulating PTEN and indirectly regulating the PI3K/AKT pathway in EEC but not in ESC of dairy goat in vitro.In addition,mi R-26 a regulated the expression of osteopontin(OPN),vascular endothelial growth factor(VEGF),Cyclooxygenase-2(COX-2)and prolactin(PRL)in endometrial cells.Therefore,we could get a conclusion that miR-26 a had very complex and diverse functions in the endometrial cells during the development of endometrial receptivity in dairy goat.4.Analysis of the differentially expressed mRNAs in the pre-receptive(PE,D5)and receptive(RE,D15)endometrium.In this study,the endometrium from gestational day 5(D5,PE)and gestational day 15(D15,RE)dairy goat were selected to systematically analyze the transcriptome using strand-specific Ribo-Zero RNA-Seq.A total of 810 mRNAs were differentially expressed genes between the RE and PE meeting the criteria of P < 0.05.Bioinformatics analysis of the DEGs revealed that a number of biological processes and pathways were potentially involved in the establishment of the RE,notably energy metabolism and a minoacid metabolism.Furthermore,we speculated that CXCL14,IGFBP3,and LGALS15 potentially participated in the development of endometrium.5.lncRNA882 acted as a competing ceRNA for miR-15 b and then regulated the level of TAB3 and LIF.In this study,a comprehensive analysis of lncRNA in goat endometrial tissues at D5(PE)and 15(RE)of pregnancy were performed by using RNA-Seq.As a result,there were 668 differentially expressed lncRNAs(DELs)were found between the PE and RE.In agreement with previous studies on human and mouse,our results indicated that the predicated lncRNAs were less in exon numbers and expression levels,shorter in length and ORF than protein coding transcripts.Further study showed that lncRNA882 could act as a ceRNA for miR-15 b what inhibited the expression of transforming growth factor-b-activated kinase 1 binding protein 3(TAB3),and then indirectly regulated the level of Leukemia Inhibitory Factor(LIF)what was helpful for the formation of endometrial receptivity in dairy goat.In conclusion,we elucidated the endometrium lncRNA profiles of PE and RE in dairy goat,and lncRNA882 acted as a competing ceRNA for miR-15 b and then indirectly regulated the level of LIF in EEC.Thus,this study helped us to better understand the molecular regulation of endometrial receptivity in dairy goat.In conclusion,the results of our present study not only provide a valuable genomic resource for the identification of differentially expressed miRNA and lnc RNA in endometrium of dairy goat,but also enrich the reference genome annotation of goat.Meanwhile,our findings accumulate experimental data in support of further research on the regulated function of miRNA and lncRNA,and provide theoretical basis for the study of molecular mechanism in the development of endometrial receptivity in dairy goat.