Study Of Interferon-τ Regulates The Expression And Function Of BoLA-Ⅰ Through Bta-miR-145/204 In Endometrium Of Early Pregnant Dairy Cattle

Interferon-tau(IFN-τ)is secreted by trophoblast cells of ruminant embryo during the early pregnancy,which is regarded as pregnancy recognition signal of ruminant.IFN-τ possesses the functions of anti-luteinolysis,anti-virus,immunomodulation and so on.Especially,it plays an important role in regulating the immunosuppressive environment in uterus during embryo implantation and providing conditions for embryo implantation.The regulation of bovine leukocyte antigen(BoLA-Ⅰ)by IFN-τ during the early pregnancy,also known as embryo implantation window is of great significance to dairy cattle.IFN-τ can regulate the immune environment of maternal and fetal interface to improve the receptivity of maternal uterus to fetus and increase the success rate of embryo implantation through regulating BoLA-Ⅰ and other pregnancy-related genes.BoLA-Ⅰ,a major histocompatibility class I(MHC-Ⅰ)related protein in cattle,is expressed in bovine embryonic trophoblast and endometrial epithelial cells(EECs),which is closely related to the maternal immune tolerance to the fetus in early pregnancy.And IFN-τ also could modulate the differential expression of miRNAs in isolated and cultured bovine endometrial epithelial cells(bEECs).miRNAs are commonly found in eukaryotes.It participates in various physiological and pathological processes by regulating the target genes,and also plays an important role in pregnancy immunity and remodeling of uterine environment.Our previous studies showed that IFN-τ could up-regulate the expression of BoLA-Ⅰduring early pregnancy in dairy cattle.And IFN-τ also could modulate the differential expression of miRNAs in bEECs.Through the preliminary deep sequencing data,it was found that some distinct differentially expressed miRNAs might be related to the regulating of BoLA-Ⅰexpression by IFN-τ in bEECs.In order to further verify the relationship between the expression of miRNAs and BoLA-Ⅰ,to provide experimental basis for further study on the immune mechanism of cow pregnancy.And to explore the potential mechanism of IFN-τ assisting embryo immune escape and providing opportunities for embryo implantation in early pregnancy,this study was begin with the verification of the target gene of miRNA,and some further studies were carried out.The main contents and results were:(1)Our preliminary deep sequencing data illustrated that the expression of bta-miRNA-145 and bta-miRNA-204 were down-regulating in bEECs with IFN-τ treatment.The endometrial tissue of early pregnant dairy cattle was collected during the implantation for experience in vivo.The results of q PCR and Western blot showed that the m RNA level of bta-miR-145 and bta-miR-204 in endometrium of early pregnancy cows was significantly lower than that in non-pregnant cows,but the expression of MIC1 and BoLA was up-regulating obviously,and the protein level of BoLA-Ⅰ was immensely increased in early pregnancy cows.(2)The primary bEECs were isolated and cultured for in vitro validation.The results shown that with the treatment of IFN-τ(200 ng/m L),the m RNA level of bta-miR-145 and bta-miR-204 were decreased sharply but the expression of MIC1,BoLA and BoLA-Ⅰ was increased enormously compared with the control group.(3)The base pairing of bta-miR-145 and MIC1 3’UTR target sequence,bta-miR-204 and BoLA 3’UTR target sequence were analysis by bioinformatics technique.And the the main bioinformatics indexes,such as the secondary structure and free energy of miRNA combined with target gene to form RNA double strand were analyzed.It was preliminarily determined that MIC1 was the target gene of bta-miR-145,BoLA was the target gene of bta-miR-204.Then the results of duel-luciferase reporter assay shown,bta-miR-145 could significantly inhibit the activity of MIC1 3’UTR,while bta-miR-204 could immensely inhibit the activity of BoLA 3’UTR.It was further confirmed that MIC1 might be the target gene of bta-miR-145 and BoLA might be the target gene of bta-miR-204.(4)The over-expression and inhibition of bta-miR-145 and bta-miR-204 showed that transfection with bta-miR-145 mimics could significantly inhibit the m RNA expression of MIC1,while the m RNA expression of MIC1 was enormously up-regulated after transfection with bta-miR-145 inhibitor in bEECs.With the transfection of bta-miR-204 mimic,the m RNA expression of BoLA was immensely down-regulated,while the m RNA expression of BoLA was sharply up-regulated after transfection with bta-miR-145 inhibitor in bEECs.With the IFN-τ treatment,the m RNA level of MIC1 and BoLA were both increased dramatically,and the protein levels of BoLA-Ⅰ was significantly increased too.It was further confirmed that MIC1 was the target gene of bta-miR-145,and BoLA was the target gene of bta-miR-204.And IFN-τ could mediate the expression of BoLA-Ⅰ through regulating the target genes of bta-miR-145 and bta-miR-204.(5)In order to validate the effect of IFN-τ on extracellular matrix and immune microenvironment during embryo implantation,the m RNA levels of ADAM 10,ADAM 17,PD-L1 and PD-L2 in endometrium of non-pregnancy and early pregnancy cows were analyzed by q PCR.The results indicated that the m RNA levels of ADAM 10,ADAM 17,PD-L1 and PD-L2 were increased to varying degrees in the endometrium of the early pregnancy.IFN-τ could significantly increased the expression of ADAM 10,ADAM 17,PD-L1 and PD-L2 in bEECs.(6)In order to verify whether IFN-τ regulates the expression of ADAM 10 and ADAM 17 through the regulating of MIC1 and regulates the expression of PD-L1 and PD-L2 through the regulating of BoLA,si RNA interference technique was performed in bEECs.The results showed that the m RNA levels of ADAM 10 and ADAM 17 were enormously down-regulated after transfection of MIC1 si RNA in bEECs.And the m RNA levels of PD-L1 and PD-L2 were significantly down-regulated after transfection of BoLA si RNA.It demonstrated that IFN-τ might regulate the expression of ADAM 10 and ADAM 17 through the regulation of MIC1,and regulate the expression of PD-L1 and PD-L2 through the regulation of BoLA.IFN-τ might affect embryo implantation via monitoring ECM and immune tolerance by regulating ADAM 10,ADAM 17,PD-L1 and PD-L2.(7)To further analyze the effect of IFN-τ on embryo implantation,a model of mouse embryo implantation failure induced by LPS was established.The results showed that IFN-τ could increase the number of implanted embryos in LPS-treated pregnant mice.IFN-τ could inhibit the expression of several inflammatory cytokines(IL-1β and TNF-α)induced by LPS and upregulate the expression of anti-inflammatory cytokine IL-10.In conclusion:(1)MIC1 is the target gene of bta-miR-145,BoLA is the target gene of bta-miR-204.(2)IFN-τ increases the expression of ADAM 10 and ADAM 17 by inhibiting bta-miR-145 to up-regulate the expression of MIC1 in order to influence the ECM and promote the process blastocyst adhesion and invasion.(3)IFN-τ increases the expression of PD-L1 and PD-L2 by inhibiting bta-miR-204 to up-regulate the expression of BoLA,thus affecting the immune microenvironment of the maternal and fetal interface and promoting the fetal immune escape.(4)IFN-τ increases the success rate of embryo implantation via up-regulating the expression of BoLA-Ⅰrelated factors by down-regulating the expression of bta-miR-145 and bta-miR-204.(5)IFN-τ increases the number of embryo implantation induced by LPS through the regulation of immune balance in mouse...