| Bovine reproductive disorders mostly occur during early pregnancy,causing huge economic losses to related industries.The study of the uterine immune system in the early pregnancy of dairy cows provides insight for dairy cow industry.During this period,the endometrium presents a certain level of immune response.NF-κB is activated,and expression levels of downstream COX-2,cytokines,etc.increase,which are considered to display multiple functions such as endometrial remodelling,placenta formation,or protecting body from the invasion of pathogens.However,the uterine immune during early pregnancy in cows maintains relatively balanced throughout this period,for usually an over-activating immune response causes inflammation,becoming a main cause of pregnancy failure.Progesterone is among hormones that have significant changes in blood concentration during early pregnancy in dairy cows.The concentration in blood reach about 10 ng/m L in Day 45 after gestation.Studies have also shown that progesterone could stimulate bovine endometrial epithelial cells(b EECs)to produce TNF-α and IL-1β,etc.cytokines.However,how progesterone induces the immune response of b EECs in early pregnancy and the corresponding regulatory mechanism need to be further elucidated.Nuclear receptor coactivator 3(NCOA3)is the co-activation factor entwined with transcription factors(such as NF-κB,AP-1 and p53)and progesterone receptor.NCOA3 is closely related to embryo implantation,placenta formation and other reproductive processes,and it is highly expressed in uterus during early pregnancy.mi RNAs regulate a variety of diseases and physiological processes in vivo by targeting the 3’-Untranslated Region(3’-UTR)of target genes to silence the expression.In this experiment,Traget Scan prediction was first performed to select mi R-20 b which characterised as targeting NCOA3 and having high scores,then the uterine tissues of early-pregnant(Day 60)and non-pregnant(Day 0)dairy cows were harvested to reveal the tendency of changes in endometrial microenvironment,NCOA3 and mi R-20 b.b EECs were used as in vitro objects to reveal relations between progesteroneinduced immune response and NCOA3/NF-κB axis,confirming the binding potential between mi R-20 b and NCOA3,and determining whether high expression level of mi R-20 b served as the regulatory molecule in endometrium immune.The above studies provide a theoretical basis for elucidating the regulation mechanism of the endometrial immune microenvironment of dairy cows during early pregnancy.Critical results and conclusions are below:1.The endometrium in bovine early pregnancy(Day 60)presented a certain level of immune response.H&E results showed that the uterine glands of dairy cows were intact in early pregnancy,and there were no obvious pathological changes such as rupture and fragmentation.From IHC and RT-q PCR results,TNF-α and IL-1β and COX-2expressions in Day 60 group were significantly higher than those of non-pregnant counterparts(p < 0.05).NCOA3 m RNA and protein expression significantly increased in the early-pregnancy uterine tissues(p < 0.01).2.Progesterone induced immune response by targeting NCOA3/NF-κB axis in b EECs.CCK8 test results showed that progesterone had no significant effects on the cell viability of b EECs with the final concentration no more than 50 ng/m L,but to inhibit significantly the cell viability in 100 ng/m L group(p < 0.05).When b EECs were stimulated by 10 ng/m L progesterone,m RNA and protein levels of NCOA3 were significantly up-regulated at 12 h(p < 0.05).The expressions of p-IκBα and p-p65 proteins in the NF-κB signaling pathway gradually increased with the prolonged stimulation of progesterone.The expression levels of TNF-α,IL-1β m RNA and COX-2protein,which were downstream genes of NF-κB,were significantly up-regulated at 12 h(p < 0.01).That progesterone activated the expression of cytokines and COX-2 in b EECs through the NCOA3/NF-κB axis was demonstrated by transfecting si RNA in b EECs to silence the expression of NCOA3.The experimental results showed that after silencing NCOA3,progesterone-induced TNF-α and IL-1β m RNA level and protein secretion level were significantly inhibited(p < 0.05).COX-2 protein level was significantly downregulated(p < 0.05),and the phosphorylation level of IκBα and p65 was significantly inhibited(p < 0.05).3.mi R-20 b regulated progesterone-induced immune response in b EECs by targeting NCOA3.mi R-20 b expression increased significantly in early-pregnant uterine tissues during,compared with non-pregnant counterpart(p < 0.05).When b EECs were stimulated by various progesterone concentrations for 12 h,NCOA3 were found evidently up-regulated in 10 ng/m L group(p < 0.05).When b EECs were stimulated by 10 ng/m L progesterone with various time periods,NCOA3 were found evidently up-regulated at the8 h and 12 h time point(p < 0.05).Target Scan predicted the binding relationship between mi R-20 b and NCOA3.Dual luciferase assay further confirmed that mi R-20 b mimics could significantly down-regulate the luciferase activity of wild-type NCOA3 3’-UTR vector(p < 0.01).Overexpression of mi R-20 b in b EECs could significantly inhibit the protein expression of NCOA3(p < 0.05),while silencing endogenous mi R-20 b could promote the protein level of NCOA3(p < 0.05).When treated with mi R-20 b mimics,there was a decrease in progesterone-induced TNF-α and IL-1β m RNA expression and secretion levels,COX-2 protein level,and phosphorylation level of IκBα and p65(p <0.05).The results showed that higher level of progesterone induced higher expression of mi R-20 b in early pregnancy cows.mi R-20 b blocked NF-κB signaling pathway by targeting NCOA3,therefore inhibiting the immune response of endometrial epithelial cells induced by progesterone.This process might be a self-regulatory mechanism in maintaining uterine immune balance for early-pregnant cows,and may be related to the success of reproductive processes such as uterine tissue remodelling,alternation of uterine immune and placenta formation. |
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