Research On The Regulatory Mechanism Of Circ-8073 And MiR-449/34 Family In The Establishment Of Receptive Endometrium In Dairy Goats

In the reproductive process of female livestock,receptive endometrium is one of the crucial and indispensable conditions for successful implantation of the embryo.The impaired receptive endometrium is an important reason for the failure of embryo implantation,which will cause the major economic loss in stockbreeding.Accumulated studies found that non-coding RNAs(ncRNAs)regulate gene expression at the post-transcriptional level,and played key regulatory roles in many physiological processes,including the establishment of receptive endometrium.Our previous studies showed that miRNAs played important roles on the establishment of receptive endometrium in dairy goats.In addition,circRNAs could act as competitive endogenous RNAs(ceRNAs)to release the inhibitory effect of miRNAs,thereby target regulating genes at the post-transcriptional levels.However,the regulatory function and mechanisms of circRNAs on the establishment of receptive endometrium in dairy goats remain largely unknown.In this study,the differentially expressed circRNAs were analyzed in receptive(RE,D15)and pre-receptive(PE,D5)endometrium of dairy goats using High-throughput sequencing technology.RNA interference(siRNA),Overexpression,Dual luciferase gene reporter system,In situ hybridization,Immunocytochemistry,Scanning electron, EdU,Western Blot and other techniques were used to explore the regulatory mechanism of circRNA,miRNA and mRNA on endometrial epithelial cells(EECs)and the establishment of receptive endometrium in dairy goats.The main results of this study are as follows:1.Identification of circ-8073 in receptive endometrium of dairy goats,and it function as a ceRNA to promote EECs proliferation by adsorbing miR-449aIn this study,RNA-seq was performed on receptive and pre-receptive endometrial tissues of dairy goats,and a total of 334 circRNAs were found differential expressed between the PE and RE.And 30 circRNAs were co-expressed in the two physiological stages,whereas 65 circRNAs were specifically expressed in the RE and 239 circRNAs were specifically expressed in the PE.Circ-8073(circRNA8073)specifically expressed in RE and had the highest expression level.These results suggested that circ-8073 might play an important regulatory role in the establishment of receptive endometrium in dairy goats,so circ-8073 was selected as the research object in this study.In addition,circ-8073 not only had the same expression tendency to its host gene CRIM1 in the endometrium tissues of dairy goats,but also could function as a sponge for miR-449 a,and inhibited the activity of miR-449 a to promote EECs proliferation.2.miR-449/34 family reduced EECs apoptosis by binding to circ-8073MiR-449/34 family(including miR-449 a,miR-34 a and miR-34c)were widely expressed in various tissues of dairy goats,and their expression levels in RE were significantly up-regulated,and induced EECs apoptosis.In addition,miR-34 a and miR-34 c could up-regulate the expression levels of miR-449 a,and there was a positive feedback regulation between miR-34 a and miR-34 c,but miR-449 a did not affect miR-34a/34 c in EECs.In addition,circ-8073 could adsorb miR-34 a and miR-34 c through "molecular sponge" and had a negative regulatory relationship with miR-34 a or miR-34 c in EECs..3.The regulatory mechanism of the target gene CEP55 of miR-449/34 family in EECsIn this study,RNA-seq was used to screen the target genes of miR-449 a in EECs.And a total of 59 differential expression genes(DEGs),including 7 down-regulated genes and 52 up-regulated genes,were obtained in the miR-449 a mimic group compared with the NC group.Further identification revealed that CEP55(Centrosomal protein 55)was a common target gene of miR-449 a,miR-34 a and miR-34 c,and all these miRNAs down-regulated the expression level of CEP55 in EECs.The expression level of CEP55 markedly decreased in RE compared to PE,especially in EECs.In addition,circ-8073 up-regulated the expression level of CEP55,but CEP55 down-regulated circ-8073.This result indicated that circ-8073,miR-449/34 family and CEP55 formed a feedback regulation network in EECs.Furthermore,CEP55 promoted EECs proliferation.The above results indicated that circ-8073 functioned as a ceRNA to regulate the CEP55 expression level by sponging miR-449/34 family,thereby promoting EECs proliferation.4.CEP55-FOXM1-VEGFA participated in the establishment of receptive endometrium in dairy goatsThe expression levels of FOXM1(Forkhead box protein M1)and VEGFA(Vascular endothelial growth factor A)significantly increased in RE compared to PE,and FOXM1 up-regulated the expression level of VEGFA and participated in the establishment of receptive endometrium.In addition,FOXM1 promoted EECs proliferation though the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways what mediated by VEGFA-VEGFR2.This study showed that there might be a negative regulatory relation between CEP55 and FOXM1 in EECs,and CEP55 could reduce VEGFA level.The above results suggested that CEP55 could be involved in the regulation of the establishment of receptive endometrium in dairy goats.5.The regulatory mechanism of circ-8073 and miR-449/34 family on the establishment of receptive endometriumCirc-8073 participated in the establishment of receptive endometrium by up-regulating the expression levels of FOXM1 and VEGFA proteins in EECs.Furthermore,circ-8073 could active the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways in EECs,but miR-449/34 family blocked the two signaling pathways.And there were negative feedback loops and cross-talks between PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways in EECs.Further research found that miR-449/34 family could participate in the development of receptive endometrium by regulating the protein levels of VEGF,FOXM1 and OPN(Osteopontin).In addition,miR-449 a could contribute to the establishment of receptive endometrium in mice in vitro.In summary,this study screened and identified that circ-8073 specifically expressed in receptive endometrium of dairy goats,and preliminarily revealed that circ-8073 functioned as a ceRNA coordinated with miR-449/34 family and CEP55-FOXM1-VEGFA to participate in the establishment of receptive endometrium in dairy goats at the circRNA,miRNA and mRNA levels.In addition,this study not only provides a basis for the further research on molecular regulation mechanism of the establishment of receptive endometrium,but also has important theoretical guiding significance for the research on improving the conception rate and important practical significance for promoting the industrial development of dairy goats.