The Expression Of Z Chromosome Linked MiRNAs In Chicken Embryos Of Two Sexes At The Early Stages And The Functional Analysis Of MiR-2954

Cell autonomous sex identity theory consider that the dosage of genes in Z chromosome determine the sex of chicken, although the exact gene is unknown.Many studies indicate that miRNAs on sex chromosome play important roles on mammalian reproductive development. Therefore, we speculate that Z chromosome linked miRNA may be crucial in the gonad development and sex determination. On the base of our former study, 10 of Z chromosome linked mi RNAs were selected to measure their expression profiles in the early embryo(incubated for 2-8.5d) using Real-time Quantitative PCR, and then the expression of male-biased miR-2954 in eight tissues of E10.5 chicken embryo was detected. Further the regulatory relations between miR-2954 and its target genes were studied through the dual luciferase reporter system and overexpression and suppression of miR-2954. The results were listed as follow.1. The temporal expression profiles of 10 Z chromosome linked mi RNAs were detected in the early stages of female and male chicken embryos or gonads. The results showed that 7 of 10 miRNAs(miR-2954, novel-miR-66, novel-miR-37 etc.) were higher expressed in male; the expression of miR-122-5p was higher in mals at E2.5-E4.5 and higher in female in E5.5-E8.5 gonads; miR-2131-5p was expressed at significantly higher level in E3-E6.5 females. Only the expression of miR-31-3p was not regular in chicken embryo.2. The results of miR-2954 expression profiles showed the significantly or extremely significantly higher expression in males was in all detected stages of chicken embryos or gonads and all detected tissues of E10.5 chicken embryo.3. YY1 and FOXD1 genes were preliminarily chosen as target genes of mi R-2954 by comprehensive analysis of TargetScan and miRDB prediction, function analysis of target genes and structure analysis. And the targets were validated by dual luciferase reporter assay, the results showed that the duel-luciferase activity of report protein was significantly decrease after cotransfection with miR-2954, which could preliminary suggest that YY1 and FOXD1 are the target genes of miR-2954.4. The expression of YY1 gene in chicken embryos and gonads at E2-8.5 and eight tissues of E10.5 chicken embryo was detected using Real-time Quantitative PCR. Results showed that the expression of YY1 is higher in females but not significant, and its expression trend was opposite to that of miR-2954.5. The expression of YY1 was downregulated by miR-2954 both on the mRNA level and protein level detected by Real-time Quantitative PCR and Western Blot after mi R-2954 overexpression and inhibition.