IFN-τ调控奶牛子宫内膜上皮细胞差异表达BoLA-I的分子机制

BoLA-I take major contribution to maternal immune response to the semiallogenic fetus.Embryo trophoblastic cells only express IFN-τ at the period of preimplantation, and its receptor is located on endometrial epitherial cells(EEC). IFN-τ differentially mediates the expression of BoLA-I on EEC, and then regulate the immune response on the interface of maternal-fetus,and therefore make the formation of endometrial condition for implantation. To test the effect of miRNA and the upstream miRNAs/mRNAs on the differential expression of BoLA-I on EEC mediated with IFN-τ, we construct the in vitro model of IFN-τ regulating the differential expression of BoLA-I on EEC. Based on the theory of miRNA/mRNA,Illumina deep sequensing will be adapted to analyze the miRNAs spectrum on the above cell model. qRT-PCR, Northern blot,in situ hydridization will be used for the confirmation of miRNAs. Bioimfornatics will applied to predict the miRNA/mRNA what is identified with miRNA/mRNA compound analytic precess and pGL3 Luciferase report genetic carrier. Antisense oligonucleotid and miRNA overexpression methods will be adapted to test the function of special miRNA.miRNA interference and overexpression induced with retrovirus will be used to analyze the regulating effect of upstream genes ahead BoLA-I. This project will discove and identify the miRNA spectrum and the upstream miRNAs/mRNAS between IFN-τ with BoLA-I in EEC of dairy cows, and then illuminate the molecular mechanism of IFN-τ differentially regulating BoLA-I expression, which should be essential to promote the productivity of dairy cows.

BoLA-I在牛妊娠免疫过程中起决定作用。IFN-τ在围着床期由胎盘滋养层细胞分泌，调节EEC差异表达BoLA-I，继而调节母-胎界面免疫。本项目构建IFN-τ调节奶牛子宫内膜上皮细胞差异表达BoLA-I的体外细胞模型，采用Illumina深度测序技术分析EEC中差异表达的miRNA并构建miRNA表达谱及其靶向关系，利用qRT-PCR、Northern blot、原位杂交、生物信息学、miRNA-mRNA复合物、反义寡核苷酸、超表达技术、报告基因等技术鉴定和验证靶向关系及其功能；利用超表达和RNAi技术研究上游miRNA/mRNA功能，综合判断miRNA及上游miRNA/mRNA在EEC差异表达BoLA-I中的作用。项目将发现并验证相关miRNA表达谱和BoLA-I上游miRNAs/mRANs及其功能，阐明IFN-τ调节EEC差异表达BoLA-I的分子机理，为提升奶牛繁殖力提供科学依据。

BoLA-I在奶牛妊娠免疫过程中起决定作用。我们先前的研究已经证实，由胚胎滋养层细胞分泌的IFN-τ能够作用于子宫内膜上皮细胞，并且使得上皮细胞BoLA-I表达显著上升，继而调节母-胎界面免疫反应影响胚胎着床过程。然而，IFN-τ调控EEC差异表达BoLA-I的分子机制还不清楚。本项目建立在实验室成熟的原代奶牛子宫内膜上皮细胞平台上，采用高通量测序技术分析在一定浓度的IFN-τ作用下，EEC差异表达的mRNA和miRNA数目以探讨奶牛妊娠早期中IFN-τ对子宫内膜差异表达BoLA-I的分子通路及其免疫机制。同时，该项目随后也探讨了BoLA-I重链在机体先天免疫反应中的非经典功能，进一步阐明了BoLA-I分子在妊娠与免疫反应中的关键作用。. 结论：(1) 通过高通量测序，筛选了一部分差异表达的基因和miRNAs。通过GO和KEGG分析发现，差异表达的基因和miRNAs主要富集在与免疫相关的通路中，例如抗原处理与呈递等，表明IFN-τ确实扮演了一个免疫调控的角色。最后一系列差异表达的基因（RAC2, DVL3, PSMB9, STAT1, ISG15, JAK1和MUC1等）和miRNAs（miR-148b，miR-152和miR-34c 等）被验证。 (2) 在体内外模型中， 我们证实了IFN-τ可能是通过上调STAT1和STAT3磷酸化水平以及下调TRAF3水平进而调节BoLA-Ⅰ表达。(3) 在子宫内膜上皮细胞上， IFN-τ通过 bta-miR-148b/152 调控BoLA-I 重链的表达。在随后的研究中我们进一步证实了由 IFN-τ上调的BoLA-I 重链能够通过Fps-SHP-2通路负向调控TLR4介导的天然免疫反应。