The Role And Mechanism Of MiRNA In URSA By Regulating DC Differentiation Via Targeting STAT Signaling Pathway

BackgroundRecurrent spontaneous abortion?RSA?refers to the occurrence of two or more spontaneous abortions before 20 weeks of gestation.It is a pregnancy complication that occurs in about 3%of pregnant couples.Although various causes are considered risk factors for RSA,such as anatomical abnormalities in the reproductive tract,abnormal uterine structure,abnormal chromosomes in parents,endocrine and/or metabolic disorders,autoimmune abnormalities and environmental factors,the causative factors in approximately 50%of RSA patients still remains unclear and that group is defined as unexplained RSA?URSA?.URSA seriously affects reproductive health of women of childbearing age,and there is increasing evidence that maternal-fetal immune tolerance mechanism plays an important role in the progress of pregnancy.The pathogenesis of maternal-fetal immune tolerance in URSA has become a hot spot in reproductive immunology research.Dendritic cell?DC?,the unique antigen-presenting cells?APCs?,are situated at the interface between the innate and the adaptive immune system.Therefore,they are critically involved in the initiation or suppression of adaptive immune response and also proposed to be pivotal players in the decision whether the fetus is tolerated or rejected.Recent studies have shown that DC plays an important role in regulating T cell proliferation and differentiation for inducing and maintaining immune tolerance,in addition to positively enhancing the adaptive immune response of T cells.DC is believed to endow the maternal-fetal tolerance and plays a crucial role in ensuring the success of pregnancy.At present,the role of DC subsets differentiation in URSA is still unclear,and the mechanism of regulating DC subsets differentiation is still lacking.This aspect of in-depth exploration is expected to become a breakthrough in the prevention and treatment of URSA.As important members of STATs family,the signal transducer and activator of transcription 3 and 5B?STAT3 and STAT5B?,which play a key role in the development,differentiation and functional regulation of DC.The differentiation state and function of DC subsets is caused by the changes of two signaling pathway and involves in the occurrence of inflammation,tumor,allergies and other diseases.However,the role of regulating the differentiation of DC subsets in URSA is ill-defined.The further exploration in this field is expected to further reveal the pathogenesis of URSA to provide a new target and approach for treatment.MicroRNA?MiRNA?,a class of conservative,endogenous small non-coding RNAs of19-24 nucleotides in length,play critical regulatory roles in a wide range of biological and pathological processes by mainly binding to 3'-untranslated region?UTR?for post-transcriptional regulation and degradation,or inhibiting the translation of the target gene protein at the translation level to achieve the regulation of the target genes in recent years.An increasing number of studies had demonstrated that miRNAs are expressed abundantly in the human blood,decidua,villi and dysregulation of miRNAs have been associated with RSA pathogenesis.However,the role and mechanism of the miRNA-targeting STAT3/5B signaling pathway in the control of the differentiation of the DC subsets in the URSA is not yet known.It is expected that the in-depth exploration in this field will explain the pathogenesis of URSA from the perspective of epigenetic regulation.ObjectiveThe role of STAT3/5B signaling pathway in regulating the differentiation of DC subsets in URSA and the mechanism of miRNA targeting regulation are investigated,then the pathogenesis of URSA is explained,which will provide a new target and approach for clinical diagnosis and treatment of URSA from the perspective of epigenetics.MethodsThe peripheral blood mononuclear cell?PBMC?and decidual tissue of patients were collected from 30 cases of normal pregnant?NP?women and 30 cases of URSA,and then divided into control group and URSA group.The differentiation state of the DC was detected by flow cytometry in PBMC and decidual tissue.The expression level of STAT3 and STAT5B mRNAs as well as related transcription factors E_2-2-2 and ID₂ mRNAs was detected by q-PCR in PBMC and decidual tissue.MiRNAs of differential expression from PBMC between NP group and URSA group were detected by high-throughput miRNA microarray.MiRNAs of differential expression which may regulate STAT3 and STAT5B were predicted by bioinformatics software.MiRNAs of differential expression were verified by q-PCR,and the influence of the miRNAs of differential expression on STAT3 and STAT5B signaling pathway was observed.The dual-luciferase reporter gene was used to determine the binding sites of the miRNAs to the STAT3 and STAT5B.Results1.Compared with the control group,the proportion of plasmacytoid DC?pDC,CD11c^-CD123⁺?was significantly lower?P<0.05?,while the proportion of conventional DC?cDC,CD11c⁺CD123^-?was markedly higher?P<0.05?in PBMC and decidua of URSA,suggesting that the changes of the DC subsets played an important role in URSA.2.Compared with the control group,the mRNA expression of STAT3 and E_2-2 was significantly lower?P<0.05?,while the mRNA expression of STAT5B and ID₂ was increased markedly?P<0.05?in PBMC and decidua of URSA,suggesting that STAT3 and STAT5B signaling pathway played an important role in URSA.3.The predicted results of the high-throughput miRNA microarray analysis and the bioinformatics software showed that the STAT3 mRNA 3'UTR contains two predicted target sites for miR-6875-5p;STAT5B mRNA 3'UTR contains one predicted target sites for miR-23a-3p.4.The expression of miR-6875-5p was remarkably increased?P<0.05?,while the expression of miR-23a-3p was significantly decreased?P<0.05?in PBMC and decidua of URSA.5.Pearson correlation analysis showed that the expression of miR-6875-5p was significantly negatively correlated with the mRNA expression of the STAT3 and E_2-2?P<0.05?;the expression of miR-23a-3p was also markedly negatively correlated with the mRNA expression of STAT5B and ID₂?P<0.05?.6.The upregulated expression of miR-6875-5p remarkably decreased the mRNA expression of STAT3 and E_2-2?P<0.05?as well as the total protein expression and phosphorylation level of STAT3;the downregulated expression of miR-6875-5p remarkably increased the mRNA expression of STAT3 and E_2-2?P<0.05?as well as the total protein expression and phosphorylation level of STAT3.7.The upregulated expression of miR-23a-3p markedly decreased the mRNA expression of STAT5B and ID₂?P<0.05?as well as the total protein expression level of STAT5B;the downregulated expression of miR-23a-3p markedly increased the mRNA expression of STAT5B and ID₂?P<0.05?as well as the total protein expression level of STAT5B.8.The dual-luciferase reporter gene confirmed that miR-6875-5p binds STAT3 mRNA3'UTR directly by the principle of complementary base;miR-23a-3p binds STAT5B mRNA3'UTR directly by the principle of complementary base.Conclusion1.The proportional shifting of DC subsets may be regulated by STAT signaling pathway?the decreasing proportion of pDC,the increasing proportion of cDC?is involved in the occurrence of URSA.2.The increased expression of miR-6875-5p inhibits STAT3 signaling pathway and further decreases the differentiation of pDC subsets,which may be an important cause of URSA.3.The decreased expression of miR-23a-3p promotes STAT5B signaling pathway and then increases the differentiation of cDC subsets,which may be an important reason for the occurrence of URSA.4.Epigenetic immune regulation of miRNAs participates in the occurrence of URSA,and targeting miRNAs regulates STAT signaling pathway to reverse the imbalance of DC subsets,which may be an effective target and pathway for the prevention and treatment of URSA.