The Role Of Chicken MiR-1b-3p In Follicular Theca Cells And Analysis Of Polymorphism In The Promoter Region

In poultry,ovary is one of the most significant reproduction organs of the hens,and it is closely related to the production performance,so the biological function of the ovary is widely concerned.The development of the ovary is not only affected by environmental and nutritional factors,but also regulated by the hypothalamic-pituitary-gonad-axis(HPGA).HPGA plays a significant role in the development of ovary by affecting the production of steroid hormones.In our previous study,we performed on the change of chicken hypothalamus and ovaries before or after sexual maturation by RNA-Seq.The results revealed that the expression of miR-1b-3p in hypothalamus and ovary rose significantly after sexual maturation and suggested that miR-1b-3p may be involved in the regulation of chicken sexual maturation and ovarian development.In this study,the expression and function of miR-1b-3p and the polymorphism of its promoter region was investigated.The main results are as follows:(1)The expression of 30 miRNAs was no significant change in hypothalamus of Jining Bairi chicken before sexual mature(60 days,100 days and 140days),but a significant increase expression was detected after sexual mature.In follicles,the expression of miR-1b-3p was the highest in the theca cells of hierarchical follicles.(2)MiR-1b-3p mimics were transiently transfected into the hierarchical follicular theca cells,we found that miR-1b-3p could significantly increase the expression of MAPK1(P<0.05)and IGF1(P<0.05).(3)CCK-8 assay showed that miR-1b-3p could promote the proliferation of chicken hierarchical follicular theca cells.(4)An effective single nucleotide polymorphism(SNP): rs737028527(G>A)was identified by analyzing the sequence of promoter region.According to the dual luciferase reporter system and the qRT-PCR,the G allele could promote the activation and expression of miR-1b-3p significantly.The association analysis between genotype of rs737028527 egg laying traits was analyzed in White Recessive Rock,the results showed that hens with AA genotype laying in earlier days and have more number of laying eggs up to 48 weeks.The transcription factor Sp1 might be combined with the G allele promoter to promote the promoter activity of miR-1b-3p(P<0.05).In summary,the expression and function of miR-1b-3p in follicular cells of chicken were investigated.The role of miR-1b-3p in chicken follicular cells is preliminarily described.Analysis of miR-1b-3p promoter region revealed that an effective SNP locus influenced the expression of miR-1b-3p and was associated with egg laying traits by altering the binding of transcription factor Sp1.