| As a new type of cell communication medium,extracellular vesicles can influence target cells/tissues on the function and phenotype,which could carry miRNA,protein and other molecules.Studies have shown that maternal plasma EVs miRNAs play a significant role in maternal-fetal communication,pregnancy recognition,embryo attachment and pregnancy diagnosis,and can mediate the regulation of uterine immune microenvironment in early pregnancy.At present,the expression of miRNA in maternal plasma and plasma EVs of goats during different gestation periods and its regulation mechanism on uterine immune inflammation are not clear.Therefore,this study based on the laboratory prophase of miRNA sequencing data,explore the expression patterns of plasma EVs differentially expressed miRNAs in plasma EVs,plasma and uterine tissues during different pregnancy periods,as well as the expression of their target genes in uterine tissues in the early pregnancy period,so as to provide early theoretical basis for the regulation mechanism of uterine immune microenvironment in early gestation.In the study,Dazu Black Goat was selected as the research object.Firstly,EVs in maternal plasma of non-pregnant(NP),pregnant 30d(P30),pregnant 60d(P60),pregnant 90d(P90)and pregnant 120d(P120)goats were isolated and identified,and the variation rules of EVs concentration in different stages of pregnancy were analyzed.Secondly,the expression of miRNAs in plasma,plasma EVs and uterine tissue in early pregnancy was explored.Then,we detected the expression levels of miRNAs in plasma and plasma EVs during different gestation periods;finally,analyzed the expression of miRNAs and their target genes in early pregnancy uterine tissue,and the function was verified by dual luciferase report.The results are as follows:1.Expression of plasma EVs in different gestation periodsThrough the separation and identification of the EVs of NP,P30,P60,P90 and P120 in goat plasma and the detection of the concentration of EVs,it was found that the isolated EVs were spherical or round cup-shaped membrane vesicles with a diameter of 30-150 nm,and could express the EVs marker proteins CD9,CD63 and Alix;with the process of gestation,the quantity of EVs presented a significant increase;2.Expression of miRNAs in plasma,plasma EVs and uterine tissue in early pregnancyBy detecting the expression levels of miRNAs in NP and P30 goat plasma EVs,it was found that the expression levels of miR-127-3p,miR-146b-5p,miR-342-5p,miR-543-3p and miR-379-5p in P30 plasma EVs was significantly higher than that of NP(P < 0.01),and the expression level of miR-223-3p in P30 plasma EVs was significantly lower than that of NP(P < 0.01).The expression levels of miR-127-3p,miR-146b-5p and miR-223-3p in P30 plasma were significantly lower than those in NP(P < 0.01),and the expression levels of miR-342-5p,miR-543-3p and miR-379-5p in P30 plasma were significantly higher than those in NP(P < 0.01).To sum up,miR-127-3p,miR-146b-5p,miR-342-5p,miR-543-3p and miR-379-5p were significantly overexpressed in goat plasma EVs during pregnancy.We further detected the expression of these miRNAs in uterine tissues,and the results indicated that the expression levels of miR-342-5p,miR-379-5p and miR-223-3p in uterine tissues of P30 were significantly lower than those of NP(P < 0.01),and the expression levels of miR-127-3p and miR-146b-5p in uterine tissues of P30 were significantly higher than those of NP(P < 0.01),while there was no significant difference of miR-543-3p(P >0.05).3.The expression of miRNAs in plasma and plasma EVs during pregnancyWhether miRNAs are different between plasma and plasma exosomes at various gestation periods,the results demonstrated that miR-127-3p,miR-146b-5p and miR-223-3p in plasma EVs of P60 reached a significant level than those of NP,P30,P90 and P120(P < 0.01).The plasma EVs expression level of miR-342-5p in P90 was significantly higher than that in the other four periods(P<0.01),while there was no significant difference in plasma EVs of P30 and P120(P > 0.05).The expression level of miR-379-5p in plasma EVs of P120 was significantly higher than that of the other four periods(P<0.01).The expression level of miR-543-3p in the plasma EVs of P30 and P120 was significantly higher than that of the other three periods(P < 0.01),but there was no statistical difference between the plasma EVs of NP,P60 and P90(P > 0.05).The expression level of miR-342-5p in plasma of P30 and P60 was significantly higher than that of the other three periods(P< 0.01),but there was no significant difference between NP and P90(P > 0.05).The expression levels of miR-379-5p and miR-543-3p in plasma of P30 were significantly higher than those of the other four stages(P < 0.01).4.The expression of miRNAs in plasma and plasma EVs in different gestational periods were comparedBy comparing the differences of miRNAs in plasma and plasma EVs during the same gestation period,it was found that there were no significant differences in miR-127-3p,miR-342-5p and miR-379-5p(P > 0.05).The expression level of miR-146b-5p in plasma of P60 and P90 was significantly lower than that of plasma EVs(P < 0.01).The expression level of miR-223-3p in NP was significantly lower than that in plasma EVs(P < 0.01),and the expression level of miR-223-3p in P30 was significantly higher than that in plasma EVs(P < 0.05).The expression level of miR-543-3p in plasma of NP and P30 was significantly lower than that of plasma EVs(P < 0.01).5.Detection of changes in uterine tissues of miR-146b-5p and its target genes in the early pregnancyBy detecting the expression levels of miR-146b-5p and its target genes in early pregnancy,it was found that the expression level of miR-146b-5p in P30 was significantly higher than that in NP(P < 0.01),and the expression levels of target genes TRAF6,IRAK1,MMP16,CCDC6,HNRNPD and PPP1R11 in P30 were significantly lower than that in NP(P < 0.01).6.Functional verification of miR-146b-5p and target gene TRAF6Through dual luciferase reporter gene detection,it was found that compared with the negative control group,miR-146b-5p mimic significantly down-regulated the luciferase activity ratio of wild-type TRAF6 3’UTR(P < 0.01);while transfected miR-146b-5p mimic had no significant effect on the luciferase activity of mutant TRAF6 3’UTR(P < 0.05).In conclusion:(1)The results demonstrated that the quantity of EVs increased with the process of gestation.(2)Detection the expression of miRNAs in plasma and plasma EVs during different pregnancy periods,found that there are certain differences,suggesting that the miRNAs playing a role may be different.Moreover,it was preliminarily found that miR-127-3p and miR-146b-5p were highly significantly expressed in plasma EVs and uterine tissues during early gestation.It is speculated that it may play a potential regulatory role in early pregnancy.(3)Detection the changes of miR-146b-5p and its target genes in the uterine tissue in the early pregnancy,found that its target genes are mainly involved in the inflammatory regulation of the uterine immune microenvironment in the early pregnancy,and found that miR-146b-5p can target TRAF6 to play a certain regulatory role in the inflammatory balance of uterine immune microenvironment in early pregnancy. |
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