The Prediction Of IFN-τ Regulating The Expression Of BoLA-I Heavy Chain By Affecting Bta-miR-152 Etc.

Various reasons cause reproduction problem, among which the main reason is embryo implantation failure. Implantation and early embryo development are the most crucial characters of pregnant physiological process. By means of expressing of immune factors in trophoblast cells and relevant tissues, the placenta can protect the fetus from immune rejection, which acts on the maternal-fetal interface. And the immune tolerance focused on the establishment and maintenance of pregnancy is dependent on the appropriate expression of MHC-I.Bovine MHC-I called Bo LA-I, is highly polymorphic, the classical（Bo LA-Ia） and non-classical class I（Bo LA-Ib） molecules of which are expressed in trophoblast and endometrial epithelial cells in a particular way, which is closely related to pregnancy immunity. IFN-τ, as the pregnancy recognition factor, is secreted by trophoblast cells during embryo implantation in ruminants, which have a regulatory role for implantation by regulating the expression of Bo LA-I heavy chain. Micro RNAs（mi RNAs） are a class of small, non-coding single-stranded RNAs with a length of 22 nt approximately, which regulate the gene expression via complementary paring with the binding site of target m RNA. mi RNAs have critical roles in many regulation processes of cellular activity. Previous research had found that mi RNAs involved in the expression of MHC-I, but the knowledge about their regulation on the process of IFN-τ regulating the Bo LA-I expression is limited.In this study, 200ng/m L IFN-τ was cultured with endometrial epithelial cells of dairy cow, so as to find out the differentially expressed mi RNAs by high-throughput sequencing. Moreover, the target genes of differentially expressed mi RNAs were predicted, and then GO annotation and KEGG pathway analysis toward the target genes were conducted to discover the important mi RNAs that associated with the expression of Bo LA-I heavy chain affected by IFN-τ. The main results are as follows:1. Preparation and quality control of total RNAThe endometrial epithelial cells were administrated with 200ng/m L IFN-τ for 0h, 6h and 12 h and one control was set up for different times respectively, each group had two repeats. 6h and 12 h later, total RNA was extracted respectively, and the RNA quantification and qualification were detected through 1% agarose gel electrophoresis, Nanodrop spectrophotometer, Qubit 2.0 fluorometer and Agilent 2100. The results showed that all the total RNA samples were of high purity, good integrity, and without significant protein contamination, which could be applied to the construction of the c DNA library.2. mi RNA sequencing analysisThe above samples were used for construction of a c DNA library and RNA-seq sequencing to analyze mi RNAs profiles. The quality control assessment showed that their error rate was 0.01% and GC content was between 47.34% 48.53%, which were all in normal range and uniform. Overall, the obtained clean reads accounted for 97.38% 98.76% of the raw data through filtering. For further study, 18 to 35 nt small RNA sequences were chosen. Thereinto, 22 nt long sequences with high frequency（about 37.75%） at 0h, and 23 nt occupied the largest proportion（about 42.72%） at 6h and 12 h. Mapped to bovine genome by Bowtie, about 60% unique small RNA reads were mapped, which suggested that the genome annotation was relatively complete.Based on the sequence homology and conservation, 574 known mi RNAs were identified, and 109 new mi RNAs were predicted totally. Among the known mi RNAs, 338 mi RNAs were expressed in all samples with diversity. All the 109 new mi RNAs were lowly expressed, however, 14 of these mi RNAs were expressed in all samples. The expression level of bta-mi R-21-5p was the highest and mir-148 family was also moderately expressed, and overall novel₁ was highly expressed among the new mi RNAs. In addition, a total of 464 mi RNAs were differentially expressed, of which 291 were differentially expressed in experimental groups, and that 173 in control groups. Among the differentially expressed mi RNAs, bta-mi R-181 c and bta-mi R-181 d were down regulated at 6 and 12 h, and bta-mi R-152 was up regulated at 6 and 12h（except experimental group at 12h）, however the expression level in 6h experimental group was obviously lower than the control at 6h. These results suggested that IFN-τ affected the expressions of bta-mi R-152 on endometrial epithelial cells of dairy cow.Further analysis of target gene prediction, GO ontology and KEGG pathway of differentially expressed mi RNAs showed that the target genes were mainly enriched to metabolic processes and catalytic activity, etc., and were also significantly enriched to immune response and immune system process. Regarding to pathway, the target genes were enriched to some classical pathways, as well as the immune related pathways, such as allograft rejection, natural killer cell mediated cytotoxicity, and graft-versus-host disease. All the above immune related functional annotation and signaling pathways that enriched all involved Bo LA-I heavy chain. These results suggested that IFN-τ can regulate Bo LA-I heavy chain expression by affecting the expression of bta-mi R-152, and then regulate pregnancy immunity. However, the related gene and the mechanism still need to be validated and clarified.Conclusion: IFN-τ might regulate the expression of Bo LA-I heavy chain gene by affecting the expression of bta-mi R-152 etc.