Exosomal Mirna Profiling During Gestation And Its Regulatory Mechanism On The Uterine Immune Microenvironment In The First Trimester Of Dairy Cows

Reproductive disorders cause huge losses to dairy cow production,an early and accurate diagnosis of reproductive dysfunctions or aberrations are crucial to shorten the calving interval through early identification of open animals and their timely treatment and rebreeding.Therefore,exploring the biomarker associated with a normal pregnancy in dairy cows is of great significance for further understanding of the maternal-fetal immune tolerance mechanism.Exosomes have significant differences under normal or abnormal physiological conditions in terms of the cargos(nucleic acids,proteins,lipids,etc.),which indicates that exosome-based diagnosis is a very promising technology for clinical diseases.Accumulated evidence shows that placenta-derived exosomes that selectively sorted miRNAs enter the maternal blood circulation during human pregnancy.Therefore,the study of these miRNAs can theoretically reflect the fetal growth status or pregnancy progress.Based on this,exosomal molecular diagnostic technology also has broad potential in dairy cow production.Currently,research on exosomes for pregnancy in dairy cows is very limited.In this study,high-throughput sequencing technology was used to investigate the differences in miRNA enrichment in peripheral plasma-derived exosomes isolated from dairy cows under non-(gestational day,G_D 0),early-,(G_D 60),middle-,(G_D 150),and late pregnancy(G_D 240).The objective is to identify specific miRNAs in each gestation period and provide a background reference for the study of pregnancy disorders.To explore whether these exosomal miRNAs representing fetal signals are involved in maternalfetal communication,we focused on the most significantly enriched bta-miR-499 in exosomes from early pregnancy and the possible mechanism by which it regulates the maternal uterine immune microenvironment.The relevant research results and conclusions are as follows: 1.In this study,exosomes were isolated using ultra-high speed centrifugation.The morphology and particle diameter distribution of exosomes were analyzed by transmission electron microscopy(TEM)and nanoparticle tracking analyzer(NTA).Exosomes exhibited the typical structure of a cup-shaped bilayer membrane under TEM,and the particle diameter was mainly distributed between 30-140 nm.Western blot analysis of exosomal protein markers,such as CD63,CD9,and placenta-specific exosomal marker protein PLAP,showed that exosomes in all groups expressed CD63 and CD9,while only exosomes from pregnancy group expressed PLAP,indicating that placental exosomes were successfully isolated.Next,ELISA method was used to quantify the expression of PLAP in exosomes at each period of pregnancy.The results showed that the increased expression of PLAP along with pregnancy progress,which was consistent with the trend of exosomal concentration in peripheral blood.It is suggested that the increase in exosomes were mainly due to the large number of exosomes released from the pregnant placenta into the maternal blood circulation.2.High throughput sequencing technology was used to compare the differences of miRNA in exosomes isolated in the first,second and third trimester of pregnancy,and to screen out specific exosome miRNA that may exist in each stage of pregnancy,such as,bta-miR-499,bta-miR-16 a,bta-miR-20 a,bta-miR-223,and bta-miR-128 in first trimester;bta-miR-493,bta-miR-127,and bta-miR-143 in second trimester;bta-miR-122,bta-miR-182,bta-miR-183,bta-miR-200 b,and bta-miR-200 c in third trimester.It is suggested that these miRNAs may be involved in maternal-fetal communication during pregnancy.3.An appropriate immune microenvironment during early pregnancy is critical to the success of pregnancy.To investigate whether placental exosomes are involved in the change of uterine microenvironment,we first analyzed the differences of miRNA in exosomes during non-pregnancy and early pregnancy and then conducted KEGG analysis of the target genes predicted by these miRNAs.The results show that bta-miR-499 is significantly enriched in early pregnancy placenta exosomes,and these differential miRNA target genes are mainly annotated in signal pathways related to the immune-inflammatory response(p <0.05),such as tumor necrosis factor signaling pathway,MAPK signaling pathway,Rap1 signal pathway,NF-?B signal pathway,and Ras signal pathway.It is suggested that the placental exosomes may play an important role in the regulation of maternal immune microenvironment during early pregnancy,however,bta-miR-499 is one of the most potential regulators.4.Based on the possibility that the above miRNAs may be involved in the inflammatory response process,we further analyzed the expression levels of proinflammatory cytokines TNF-? and IL-6 at the distal horn of uterus in early pregnancy in cows.qPCR results showed that TNF-? and IL-6 were higher expressed in the first-trimester group than that in the non-pregnant group(p < 0.05),and immunohistochemical results also confirmed the positive expression of TNF-? and IL-6 in the first-trimester group.The above results indicated that the uterus of cows in the early pregnancy showed a biased pro-inflammatory or Th1 immune microenvironment.5.NF-?B is one of the major regulators of the inflammatory process,which can regulate the expression of downstream pro-inflammatory cytokines such as TNF-? and IL-6.Combined with KEGG analysis,the NF-?B signal may be a "switch" for regulating the uterine immune microenvironment in early pregnancy.Immunostaining showed that NF-?B was highly expressed in the uterus in the first trimester.It is suggested that NF-?B is activated in the uterus in early pregnancy and may act as a "switch" for the Th1/Th2 microenvironment in the uterus.6.LPS(1 ?g/m L)induced bovine endometrial cells(BEND)to construct a model of proinflammatory uterine microenvironment under the activation of NF-?B to investigate the regulatory potential of bta-miR-499 enriched in placental exosomes(pEXO)of early pregnancy on inflammation.Results showed that the expression of TNF-? and IL-6 were significantly up-regulated after LPS stimulated BEND cells(p < 0.05),while the trend was reversed after co-incubation of p-EXO(10 ?g/m L),but no reversal was observed in exosomes of the non-pregnant(n-EXO)group at the same doses.qPCR results showed that the expression level of bta-miR-499 in co-cultured p-EXO BEND cells was significantly increased(p < 0.05),and the expression of TNF-? and IL-6 induced by LPS was significantly decreased after transfection with bta-miR-499 mimics.The above results indicated that the placental exosome bta-miR-499 in early pregnancy has the potential to suppress the inflammatory immune response.7.To determine whether miR-499 in early pregnancy placenta exosome regulates the expression of proinflammatory cytokines by inhibiting the activation of NF-?B pathway,immunofluorescence staining of phosphorylated NF-?B p65(p-p65 showed that LPS induced NF-?B transfer from the cytoplasm to the nucleus,however,it was significantly inhibited after p-EXO incubation,while n-EXO treatment did not show obvious changes.Overexpression of the bta-miR-499(agomiR-499)showed similar results to the p-EXO treatment.Western blot analysis showed an increased level of NF-?B phosphorylation under LPS stimulation,but it was reversed by both p-EXO and overexpression of miR-499.It is suggested that p-EXO inhibits the activation of NF-?B signal through bta-miR-499,thereby regulating the expression of pro-inflammatory cytokines.8.miRNA involves in downstream biological events by inhibiting the expression of target genes.The dual-luciferase reporting assay confirmed that bta-miR-499 directly targeted the 3'UTR of Lin28 B mRNA,thereby inhibiting the intracellular Lin28 B expression level.The let-7 family has been shown to regulate the activation of NF-?B signaling in inflammatory responses,and Lin28 B has been identified as an inhibitor of let-7 family expression.To investigate whether bta-miR-499 inhibits NF-?B activation by up-regulating let-7 family expression by targeting Lin28 B.The expression of let-7 families were significantly increased after transfection of agomiR-499 into BEND cells,which was similar to that of silencing Lin28 B with siRNA.Overexpression of Lin28 B significantly inhibited the expression of let-7 families,indicating that miR-499 inhibited the expression of Lin28 B and increased the intracellular let-7 level.Subsequently,p-NF-?B reporting system and western blot analysis were used to confirm that let-7 inhibited NF-?B signal activation.qPCR results showed that a large number of let-7 enriched in p-EXO,but it was different from the members of the let-7 family that were up-regulated after p-EXO incubation,indicating that p-EXO could directly deliver let-7 to target cells,or up-regulated the intracellular let-7 level via the bta-miR-499/Lin28 B axis,thereby inhibiting the activation of NF-?B and thus reducing the expression of Th1 cytokines,and regulating the local inflammatory microenvironment.9.The mouse pregnancy model was used to further confirm the in vitro conclusions.On day 5.5 of pregnancy(the vaginal plug was found to be day 0.5 of pregnancy,E 0.5)and on day 7.5,a synthetic miR-499 inhibitor,antagomiR-499,was injected into ICR mice.(i.p.;0.5 ?mol / kg),the same amount of miR-NC was injected into the control group;Samples were collected at E8.5 and E14.5.The results showed that the number of mouse embryos significantly decreased under the intervention of miR-499 inhibitor(p < 0.05),and the average fetal weight of mice in the second trimester(E14.5)was significantly lower than that of the miR-NC group(p < 0.05),indicating that inhibiting the expression of mir-499 may change the local uterine microenvironment and results in inhibition of fetal growth.To further investigate whether the above results were caused by changes in the local inflammatory microenvironment of the uterus,immunostaining and qPCR were used to analyze the expression of NF-?B p65,TNF-?,and IL-6 in each group,respectively.Results showed that during the first trimester of the uterus,NF-?B was slightly activated and the levels of pro-inflammatory cytokines were also increased.Inhibition of miR-499 led to abnormal activation of local NF-?B and further upregulation of proinflammatory cytokines levels(p < 0.05).It is suggested that the establishment and maintenance of early pregnancy in mice relied on a moderate proinflammatory microenvironment,and with the intervention of miR-499 inhibitor,the local Th1/Th2 cytokine balance was disrupted,leading to an increased risk of pregnancy abnormalities such as embryo loss and fetal growth restriction.Conclusions:(1)miRNAs are differentially enriched by placental exosomes at different stages of pregnancy and released into maternal blood circulation.The discovery of these miRNAs provides a reference for the exploration of normal pregnancy-related biomarkers and opens a new horizon for the study of maternal-fetal communication mechanism during pregnancy;(2)Placental exosomes in early pregnancy directly or indirectly target NF-?B signaling pathways through the delivery of let-7 and miR-499 to regulate Th1/Th2 cytokine balance in the local immune microenvironment of the uterus,providing a new theoretical basis for maternal-fetal immune tolerance mechanism in dairy cows.