miRNAs介导Wnt/β-catenin信号通路调控鸡卵泡发育的分子机制

The Wnt/β-catenin signaling pathway plays an important role in the regulation of follicle growth, maturation, ovulation and luteal formation, but the molecular mechanisms that influence the expression of this signaling pathway remain unclear. Our previous study found that there are a large number of differentially expressed genes and miRNAs in the Jining Baiji chicken ovary at different developmental stages. It is noteworthy that the expression of the Wnt signaling pathway were significantly up-regulated after starting production. Meanwhile, the predicted genes of differentially expressed miRNAs were significantly enriched to Wnt/β-catenin signaling pathway, which indicated that miRNAs might mediate the Wnt/β-catenin signaling pathway to regulate ovarian follicular development in chicken. In this study, we will first verify the interactions between miRNAs and their target genes using miRNA pull-down and dual luciferase reporter genes. The overexpression and knockdown of miRNAs combined with the Top-Flash/Fop-Flash method will be used to determine the relationship between miRNAs and Wnt/catenin signaling pathway. Then, the molecular mechanism of miRNA-mediated Wnt/β-catenin pathway involved in the development of chicken follicles will be analyzed in vitro from the aspects of cell proliferation, cell apoptosis, cell cycle and hormone secretion. Finally, the functions of miRNAs would be verified in vivo. This project will illuminate the molecular mechanism that miRNA mediated Wnt/β-catenin signaling pathway to regulated follicular development in chicken.

Wnt/β-catenin信号通路在调节卵泡的生长、成熟、排卵和黄体形成过程中起重要作用，但是影响该信号通路表达的分子机制仍不清楚。我们前期研究发现，不同发育阶段的济宁百日鸡卵巢存在大量差异表达的基因和miRNA，尤其是开产后Wnt信号通路基因表达显著上调，同时差异表达的miRNA预测的靶基因也显著富集到Wnt信号通路，即miRNA可能介导Wnt信号通路调节鸡卵巢卵泡发育。本研究拟首先通过miRNA pull-down、双荧光素酶报告基因等技术验证关键miRNA与其靶基因互作，通过对miRNA上调、下调，结合Top-Flash/Fop-Flash方法确定miRNA调控Wnt/β-catenin信号通路；然后，从细胞增殖、凋亡、周期和激素分泌等角度体外解析miRNA介导Wnt通路参与鸡卵泡发育的机制；最后，对上述机制进行体内验证。研究结果将明确miRNA介导Wnt通路参与鸡卵泡发育的机制。

Wnt/β-catenin信号通路在调节鸡卵泡的生长、成熟、排卵过程中起重要作用，但是影响该信号通路表达的分子机制仍不清楚。本项目主要从3个方面开展研究：(1)验证了miR-458b-5p是否通过与CTNNB1基因结合调节Wnt/β-catenin信号通路；(2)从卵泡颗粒细胞增殖和激素分泌两个角度解析miR-458b-5p靶向CTNNB1介导Wnt/β-catenin 信号通路参与鸡卵泡发育的机制；(3)检测鉴定了miR-458b-5p及CTNNB1与鸡产蛋性状相关的分子标记。主要结果如下：（1）双荧光素酶报告试验证明CTNNB1是miR-458b-5p的直接靶点；转染、过表达和流式细胞术等系列实验表明，miR-458b-5p降低了Wnt/β-catenin通路中CTNNB1及其下游具有细胞增殖功能的CD44和MMP7的mRNA和蛋白表达，减少了等级卵中泡颗粒细胞的增殖。（2）过表达或干扰试验表明，CTNNB1基因改变卵泡颗粒细胞周期进程，促进细胞增殖；荧光共聚焦及免疫共沉淀、酵母单杂交等系列实验表明，CTNNB1是与TCF4相互作用进而调控CCND1表达从而促进细胞增殖及相关基因的表达。（3）利用上述实验技术，证明了CTNNB1与转录因子SF1相互作用并调控下游靶基因CYP11A1表达，从而促进颗粒细胞E2和P4分泌及相关基因的表达。（4）将鉴定到的miR-458b-5p及靶基因CTNNB1与鸡产蛋性状进行关联分析，筛选到10余个SNPs与产蛋性状相关。本项目基本揭示了miR-458b-5p靶向CTNNB1介导Wnt/β-catenin信号通路调节鸡卵泡颗粒细胞的增殖和性激素分泌进而调节卵泡发育的分子机制，为全面解析鸡卵泡发育的分子调控网络提供了重要补充；筛选的分子标记可为鸡产蛋性状的遗传改良提供支持，具有潜在的应用价值。