Expression Profile Of MiRNA In Different Stages Of Bovine Corpus Luteum And The Role Of MiR-378in Luteal Cells

The corpus luteum (CL) is a temporary secretory tissue that can be found infemale mammals after ovulation. It plays an important physiological function duringestrous cycle alternation, embryo implantation, and pregnancy maintenance. Thus,illustrating the mechanisms of luteal development and regression has practical valuefor controlling animal heat, ovulation, and embryo transplantation.According to recent studies, microRNA (miRNA) regulates gene expression atthe post-transcriptional level, which is a new regulative style of gene expression.miRNAs are small, non-coding endogenous RNA molecules (²2nt in length) thatbind to their mRNA targets in order to regulate the expression of target genes in asequence-dependent manner. Studies have confirmed that miRNA participates in aseries of important processes, including angiogenesis, cell proliferation, and apoptosis.The development and regression of CL also involves angiogenesis, cell proliferationand apoptosis, suggesting that miRNA may have a function in the development andregression of CL.In order to investigate the function of miRNA in the development and regressionof CL, high-throughput miRNA array was employed in the current study to profilemiRNA in bovine functional and regressed CL. Quantitative Real-Time RT-PCR wasalso used to validate the microarray data. Compared with the results obtained forbovine regressed CL,20miRNAs were up-regulated in functional CL, and14weredown-regulated, among which miR-378expression was significantly up-regulated infunctional CL (8.54-fold, p<0.01).Interferon-γ receptor1(IFNGR1)3'-UTR was predicted to contain a miR-378binding site with bioinformatics methods, which may be a target gene of miR-378.Dual-luciferase report in Hela cell indicated that miR-378can inhibit the luciferase activity of dual-luciferase recombinant plasmid with IFNGR13'-UTR. In comparison,miR-378could not affect the luciferase activity of mutant plasmid, in which seedsequences of miR-378target region was mutated. The results showed that miR-378targeted IFNGR13'-UTR (NM₀01035063.1:1608-1629nt).The expressions of miR-378and IFNGR1mRNA/protein were also detected inearly, mid-, late, and regressed bovine CL in order to explore the expression pattern ofmiR-378in the different stages of CL. The expression levels of miR-378and IFNGR1protein were reversed in four stages of bovine CL, indicating a classic miRNA-targetgene relationship.IFNGR1is a key receptor of IFN-γ, which can affect its function, while we foundmiR-378can inhibit IFNGR1expression. In order to detect the function of IFN-γ onmiR-378and IFNGR1, bovine luteal cell was isolated, and the expression levels ofmiR-378and IFNGR1mRNA/protein were respectively determined inIFN-γ-untreated and treated luteal cells. Compared with the control, IFN-γsignificantly up-regulated the miR-378and IFNGR1mRNA/protein, indicating thatmiR-378may participate in the IFN-γ induced signal pathway through IFNGR1.In this study, many differentially expressed miRNAs were found through themiRNA profiling of bovine functional and regressed CL. Target genes of miR-378were selected and validated, and the function of miR-378in bovine luteal cell waslikewise explored. All the results indicated that miRNA played an important role inluteal function, providing theoretical bases for studies on the molecular mechanism ofbovine luteal development and regression.