Targets Analysis Of MiRNA With Sexual Differentially Expression At The Early Stages Of Sexual Differentiation Of Chicken Embryo

miRNA is a family of small, non-coding RNA molecules of about 20-25 nucleotides, which regulate many biological pathways by down-regulate target gene expression at the post-transcriptional level. Some sexually dimorphic miRNA expressions during the early chicken embryos had been found in previous studies, but their target identification and function on the chicken sexual differentiation is uncertain. In order to investigate the targets regulated by miRNAs during the early stages of gonad sexual differentiation and explore the effect of miRNA on gonad development in chicken, expression patterns of miRNAs with sexual differentially expression which selected based on the early study were studied firstly by Quantitative RT-PCR and WISH methods, then their targets were analyzed by the methods combining bioinformatics with experimental validation. The results obtained were as follows:1. Temporal expression profiles of 5 miRNAs in female and male chicken embryo gonads at E3.5-6.5d were studied by Quantitative RT-PCR method, the results showed that miR-92 was expressed at significant higher level in male at all detected stages, except for E4.5d (P<0.01), and miR-2954 was with significant higher expression in male at E4.5d(P<0.01). In addition, miR-107、1767 and 31 were higher expression in female during early stage(E3.5～4.5d), but higher in male during later stage(E5.5-6.5d).2. Spatial expression pattern of miR-92 was done by WISH. The results showed that the expression signals of miR-92 in UGSs and gonads appeared higher in male than in female at E5.5d and E6.5d, and which appeared significant higher in gonads than in UGSs.3. The targets of miR-92 was predicted by TargetScan and miRDB, and ATRX was selected as the regulate target gene of miR-92 during gonad development by combining the results of functional analysis and the secondary structure and free energy of miRNA-target Dimer.4. The target predicted by bioinformatics method was validated by dual luciferase reporter assay, the result showed that the luciferase activity of report protein was significantly decrease (P<0.01) after cotransfection with miR-92, which could suggest that ATRX might be the target gene of miR-92.5. Temporal expression profiles of ATRX in female and male chicken embryo gonads at E3.5～6.5d were studied by Semi-quantitative RT-PCR, the result showed that its expression trend was opposite to that of miR-92(except E4.5d), which suggested that miR-92 might be involved in the regulation of chicken embryonic gonadal development by inhibiting the expression of its target gene ATRX.