kisspeptin通过miR-10b靶向PAI-1促进牛颗粒细胞凋亡机制研究

Our previous studies have shown that kisspeptin promotes apoptosis of bovine granulosa cell. Based on association analysis of RNA-Seq and microRNA sequencing; and dual-luciferase experiment, we put forward the hypothesis that kisspeptin promotes apoptosis of bovine granulosa cells via miR-10b targeting PAI-1. However, its mechanism is still unclear. In the present project which aims to clarify the mechanism of kisspeptin promoting apoptosis of bovine granulosa cells, transfection will be used to study the role of miR-10b and PAI-1 in the apoptosis of bovine granulosa cell. The relationship of miR-10b and PAI-1with kisspeptin will also be evaluated to test the hypothesis that kisspeptin promotes apoptosis of bovine granulosa cell via miR-10b targeting PAI-1. Secondly, core promoter of Pri-miR-10b responding to kisspeptin will be screened and its transcription factor will be searched. Binding of transcription factor to core promoter of Pri-miR-10b and its effects on expression of miR-10b will also be evaluated to reveal the mechanism of kisspeptin upregulating expression of miR-10b. Finally, techniques such as RNA-Seq will be used to study downstream of signaling pathways for PAI-1 regulating apoptosis of bovine granulosa cells. Results of this project will provide reference for full understanding of the mechanism of apoptosis in granulosa cells.

前期研究发现，kisspeptin可促进牛颗粒细胞凋亡，根据RNA-Seq和microRNA测序关联分析及双荧光素酶等试验结果，我们认为“kisspeptin通过miR-10b靶向抑制PAI-1表达而促进牛颗粒细胞凋亡”,但其机制尚不清楚。本项目拟首先利用转染等技术研究miR-10b和PAI-1对牛颗粒细胞凋亡的作用及其与kisspeptin的关系，明确kisspeptin通过miR-10b靶向PAI-1促进牛颗粒细胞凋亡；随后筛选响应kisspeptin的Pri-miR-10b核心启动子序列，寻找转录因子，研究其与启动子区的结合及对miR-10b表达的作用，揭示kisspeptin上调miR-10b表达的分子机制；最后利用RNA-Seq等技术探索PAI-1调控牛颗粒细胞凋亡的下游信号通路，阐明kisspeptin促进牛颗粒细胞凋亡的机制，研究结果为全面理解颗粒细胞凋亡调控机制提供参考。

本项目研究了kisspeptin通过miR-10b/PAI-1途径调控牛颗粒细胞凋亡的机制。我们发现，过表达miR-10b可提高牛卵巢颗粒细胞凋亡率和Caspase-3表达水平、降低Bcl-2/Bax表达比值和PAI-1表达水平；过表达PAI-1可降低牛卵巢颗粒细胞凋亡率和Caspase-3表达水平、提高Bcl-2/Bax表达比值；过表达PAI-1可抑制miR-10b诱导的颗粒细胞凋亡、提高Bcl-2/Bax mRNA表达比值、降低Caspase-3 mRNA表达水平。通过转录组测序分析，发现磷脂酰肌醇-3-激酶/蛋白激酶 B （PI3K/AKT）是PAI-1的下游途径，并通过western blot验证这一通路，使用PI3K/AKT抑制剂（Miltefosine）抑制PI3K/AKT通路，可逆转PAI-1对牛卵巢颗粒细胞凋亡的抑制作用，增强miR-10b对牛卵巢颗粒细胞凋亡的促进作用。我们还发现，过表达miR-10b可缓解kisspeptin对牛卵巢颗粒细胞凋亡的作用，过表达PAI-1可加强kisspeptin对牛卵巢颗粒细胞凋亡的调控作用。使用DNA甲基转移酶抑制剂5-AZA处理牛卵巢颗粒细胞后可显著抑制kp-10对牛卵巢颗粒细胞活力的影响，逆转kisspeptin对miR-10b的作用，表明kisspeptin可能通过DNA甲基化途径调控miR-10b表达。综上，研究结果表明，kisspeptin可通过miR-10b靶向PAI-1调控PI3K/AKT通路影响牛卵巢颗粒细胞凋亡。