Molecular Mechanism Of LncRNA FPMAL Promoting Uterine Receptivity In Early Pregnancy Of Dairy Cows By Competitive Inhibition Of FOXO1 Phosphorylation

Poor reproductive efficiency of dairy cows seriously harms the economic benefits of dairy and meat industries.Preventing early embryo loss is one of the main means to improve the reproductive rate of dairy cows.Embryo loss is closely related to maternal microenvironment,embryo quality and maternal-fetal communication.Receptive endometrium is the biological basis of embryo implantation,and the lack of receptive endometrium will result in implantation failure.Uterine receptivity in humans and rodents has been studied extensively,but histological characteristics and molecular mechanisms of uterine receptivity in dairy cows are rarely studied,and the relevant mechanisms are still unclear.The objective of this study was to investigate the histological characteristics,m RNA and lncRNA expression profiles of endometrial receptivity in cows during early pregnancy,and the molecular mechanism of receptivity.The main research results were as follows:1.Histological characteristics and receptivity biomarkers of receptive uterus in dairy cowsIn this study,the concentrations of P4 and IFN-τ in uterine samples of dairy cows were detected by ELISA to evaluate and confirm the uterine samples in early gestation.H&E staining,SEM,and TEM were used to observe the uterus tissues of early pregnancy.It was found that the endometrial lumen epithelial cells changed from columnar epithelium to cubic epithelium,pinopodes appeared on the tip of the cells,microvilli of the apical membrane became sparse,tight junction and lateral wall folds of the membrane increased,and desmosomes decreased.q PCR detection of receptivity biomarkers in early pregnancy showed that HOXA10,LIF and ITGαVβ3 were significantly up-regulated,but MUC1 was down-regulated.Epithelial biomarkers(E-cadherin)were significantly reduced,but mesenchymal biomarkers(N-cadherin)were significantly increased by WB and IF assay.The results indicated that plasma membrane transformation occurred in receptive uterus of dairy cows in early pregnancy.2.m RNA expression profile of cow receptive endometriumIn this study,libraries were constructed by ribosome removal and chain specificity,and RNA-seq were used to establish the m RNA expression profile of the receptive endometrium of dairy cows in early pregnancy.1789 m RNA differentially expressed m RNA were screened out from 22767 m RNA by fold change and significance level.Among them,1385 m RNA were up-regulated and 404 m RNA were down-regulated.GO enrichment revealed that differentially expressed m RNA clustered in cell-cell adhesion,cell-matrix adhesion,cell cycle,epithelial cell differentiation and proliferation,and cytoskeleton,etc.Further KEGG pathway enrichment analysis suggested that differentially expressed m RNA was mainly enriched in FOXO,p53,Jak-STAT,MAPK,PI3K-AKT and Wnt signaling pathways,which were closely related to cell cycle,spliceosome and EMT.AS analysis revealed a large number of AS events,which greatly enriched gene expression.These results indicated that a large number of differentially expressed m RNA was involved in the establishment of receptive uterus.FOXO1 was an important up-regulated gene that regulated the PI3K/AKT signaling pathway and was involved in cytoplasmic membrane transformation.3.lncRNA expression profile of dairy cow receptive endometriumA total of 2791 known lncRNAs and 3309 novel lncRNAs were obtained through analysis of transcripts,and 596 up-regulated lncRNAs and 165 down-regulated lncRNAs were screened.Subsequently,target genes of differentially expressed lncRNA were predicted by cis and trans.GO analysis displayed that lncRNA target genes were mainly concentrated in epithelial cell migration,cell junction,extracellular matrix binding,cell adhesion,glycoprotein biosynthesis,etc.KEGG signaling pathway analysis showed that the target genes of differentially expressed lncrnas were mainly enriched in TGF-β,Insulin,m TOR,PI3K-AKT,FOXO signaling pathways.The above differentially expressed lncRNA have various regulatory mechanisms,which provided guidance for further elucidate the molecular mechanism of uterine receptivity formation.Among them,lncFPMAL was a vital up-regulated lncRNA,which may play an important regulatory function in the formation of uterine receptivity.4.FOXO1 promoted the formation of uterine receptivity in dairy cowsWB and IF assay confirmed that FOXO1 protein was significantly increased in receptive uterus of dairy cows in early pregnancy,and mainly concentrated in nucleus.The PI3K-AKT pathway was activated in the receptive uterus.To explore the role of FOXO1 in the formation of uterine receptivity,the primary endometrial epithelial cells(b EECs)of dairy cows were isolated by enzyme digestion and mechanical method,and b EECs cell model was established in vitro by treated with E2,P4 and IFN-τ.b EECs treated with E2,P4 and IFN-τ decreased microvillus coverage,cell polarity,plasma membrane transformation and cell cycle stagnation,indicating that E2,P4 and IFN-τ induced b EECs to be transformed into receptive epithelium.However,lentivirus knockdown of FOXO1 gene significantly reduced the plasma membrane transformation,G1 phase arrest,migration and invasion of b EECs induced by E2,P4 and IFN-τ,and overexpression of FOXO1 had similar effects to E2,P4 and IFN-τ.These results indicated that FOXO1 promoted the plasma membrane transformation of b EECs and may be involved in the formation of uterine receptivity.5.Molecular mechanism of lncFPMAL’s competitive inhibition of FOXO1 phosphorylation mediated by PI3K-AKT pathway by binding to FOXO1lncFPMAL was located upstream of FOXO1 gene and its fragment size was 384 bp.RNA nucleoplasmic separation and FISH assay exhibited that lncFPMAL was distributed in both cytoplasm and nucleus.The eukaryotic expression system analysis confirmed that lncFPMAL had no protein coding ability.lncFPMAL was significantly up-regulated in the results of RNA-seq,and q PCR and FISH verified that lncFPMAL was significantly increased in receptive endometrium and b EECs induced by E2,P4,IFN-τ.Through packaging lncFPMAL sh RNA withg lentivirus and infecting b EECs,knockdown of lncFPMAL in b EECs were successfully constructed.lncFPMAL knockdown b EECs was treated with E2,P4 and IFN-τ.IF and WB detection showed that lncFPMAL knockdown prevented the decrease of E-cadherin induced by E2,P4 and IFN-τ,and inhibited the expressions of N-cadherin,Snail and Twist1,indicating that knockdown lncFPMAL was not beneficial to the transformation of plasma membrane.Flow cytometry revealed that lncFPMAL knockdown effectively eliminated E2,P4 and IFN-τ-induced G1 phase arrest,and knockdown of lncFPMAL inhibited p27 kip1 protein and increased the expressions of Cyclin D1 and Cyclin D2.In addition,knockdown of lncFPMAL significantly inhibited the migration and invasion of b EECs induced by E2,P4 and IFN-τ.IF and WB detected the cytoplasmic distribution of FOXO1 and found that overexpression lncFPMAL obviously suppressed p-FOXO1 and increased the nuclear input of FOXO1.RNA pull down confirmed that lncFPMAL could bind FOXO1 protein,and RIP analysis also reversely demonstrated this result.lncFPMAL might enhance FOXO1 transcriptional activity by interfering with AKT and SGK1-mediated FOXO1 phosphorylation by through binding to FOXO1 phosphorylation sites.In summary,the conclusions of this paper were shown below:(1)m RNA and lncRNA expression profiles were established in the receptive endometrium of cows during early pregnancy,and 1789 differentially expressed m RNA and 761 differentially expressed lncRNA were identified.(2)Although PI3K-AKT signaling pathway was activated in receptive endometrium of dairy cows,FOXO1 was still highly expressed.The mechanism was that lncFPMAL competitively inhibited FOXO1 phosphorylation mediated by PI3K-AKT pathway by binding to FOXO1,maintaining FOXO1 entry into the nucleus and transcriptional activity.(3)The high expression of FOXO1 promoted the plasma membrane transformation,proliferation inhibition,migration and invasion ability of b EECs.