miR-27a介导Wnt信号通路在牛颗粒细胞增殖分化中的作用及分子机制

Follicular development is closely related to the cow reproductive performance, and directly affect the calf production and milk production cycle. The proliferation and differentiation of granular cells are important conditions for follicular development, but the specific mechanism is still unclear. Applicant focuses on the regulation of Wnt signaling pathway in the follicular development, compositing the previous experimental results and the previous reports, we find that miR-27a and target gene DKK2 play important roles in follicular development. This study aim to construct regulation network of miR-27a and DKK2 gene in proliferation and differentiation of bovine granular cells. Through establishing bovine follicular granular cell line, the effect of miR-27a on proliferation, differentiation and hormone levels of bovine follicular granular cells will be studied. To further clarify the specific mechanism, we will study the transcriptional regulation of miR-27a, the target relationship between miR-27a and DKK2 gene and the regulation of Wnt signaling pathway in proliferation and differentiation of granular cells from molecular and cellular levels. This study aim to clarify the miR-27a-mediated Wnt signaling pathway regulate proliferation and differentiation of bovine granular cells. Parsing this scientific problem will be hopeful to providing theoretical basis and new ideas for beef cattle breeding and dairy production.

卵泡发育与母牛的繁殖性能密切相关，直接影响犊牛生产和产奶周期。颗粒细胞的增殖分化是卵泡发育的重要条件，但具体的作用机制尚不够明确。申请人专注于Wnt信号通路在卵泡发育过程中的调控作用，综合前期试验结果和文献报道发现，miR-27a与靶基因DKK2在卵泡发育过程中发挥重要调控作用，本研究旨在构建miR-27a和DKK2在牛颗粒细胞增殖分化中的表达调控网络。通过建立牛卵泡颗粒细胞系，分析研究miR-27a对颗粒细胞增殖分化和激素水平的影响；为进一步明确具体作用机制，从分子和细胞水平研究miR-27a的转录水平调控、miR-27a与DKK2靶向作用以及由此介导的Wnt信号通路在颗粒细胞增殖分化过程中的调控作用；阐明miR-27a介导Wnt信号通路调控牛颗粒细胞增殖分化的分子机制。解析该科学问题有望为肉牛繁育和奶牛生产提供理论依据和新的思路。

卵泡发育与母牛的繁殖性能密切相关，直接影响犊牛生产和产奶周期。颗粒细胞的增殖分化是卵泡发育的重要条件，但具体的作用机制尚不够明确。非编码RNA，尤其是miRNA在各种生命活动中发挥重要调控功能已经有大量的文献报道。miR-27a在家畜排卵前卵泡中表现为差异表达，提示可能参与卵泡发育的调控。申请人一直专注于非编码RNA和Wnt信号通路在卵泡发育过程中的调控作用。以此为基础，为了进一步明确miR-27a介导的调控通路对牛卵泡颗粒细胞增殖的调控功能和作用机制，我们开展了以下研究：（1）构建了动物卵泡颗粒细胞分离培养的技术平台，成功分离培养了小鼠和牛的卵泡颗粒细胞；（2）重新筛选了miR-27a的靶基因，利用信号通路分析和进一步筛选，选取Wnt 信号通路中NFAT5、VANGL1 和VANGL2基因作为拟研究对象。利用双荧光素酶报告系统检测发现，过表达miR-27a 会抑制NFAT5、VANGL1和VANGL2 的荧光活性，值得进一步研究。（3）转录因子p53通过与miR-27a启动子区2个结合位点作用抑制miR-27a的表达，miR-27a抑制靶基因NFAT5的表达水平，NFAT5能够显著促进小鼠卵泡颗粒细胞的增殖和加强Wnt的信号强度。（4）通过研究发现miR-27a能够显著抑制牛卵泡颗粒细胞的增殖，而其靶基因VANGL1和VANGL2能够显著促进牛卵泡颗粒细胞的增殖。在miR-27a启动子区筛选出发挥上调作用的转录因子Myod，miR-27a可以抑制VANGL1和VANGL2的表达水平，VANGL1和VANGL2可以相互促进对方的表达水平。综上所述，通过研究明确miR-27a及其介导的两条信号通路对卵泡颗粒细胞增殖的调控作用和机制，有望为肉牛繁育提供理论依据和新的思路。