miR-285通过Hippo信号通路调节细胞增殖与凋亡的机制研究

Hippo pathway is a highly conserved signaling network that controls organ size by restricting cell proliferation and promoting apoptosis. Our previous research demonstrated that Brahma regulated the Hippo pathway activity through forming complex with Yki-Sd (Cell.Signal, 2015). microRNAs have emerged as key regulators of cellular proliferation and apoptosis by cross-talking with Hippo pathway. However, many crucial questions await answers before the regulatory mechanism can be fully elucidated. Recently we identified miR-285 as a novel microRNA gene in Drosophila that can regulate Hippo signaling activity. Our preliminary data showed that overexpression of miR-285 in Drosophila resulted in a strong undergrowth phenotype. Furthermore, we found that overexpression of miR-285 significantly downregulate multiple downstream target genes of Hippo signaling pathway and promote apoptosis. In this project we will further study the miR-285 mediated regulatory mechanism of cellular proliferation and apoptosis through Hippo pathway. We will identify the direct target genes of miR-285 by utilizing Drosophila RNAi libraries and reporter gene system based on bioinformatic analysis. Moreover we will explore the biological role of miR-285 during Drosophila development by generating miR-285 mutant fly using CRISPR/Cas9 and imprecise transposon excision technique. This study will enrich our understanding of the regulatory network of Hippo pathway during development.

Hippo通路在调控器官生长、细胞增殖和凋亡中发挥重要作用，我们发现Brahma与Yki-Sd形成复合物，调节Hippo信号通路（Cell.Signal，2015）。miRNA作为重要调节因子可以与Hippo通路交互作用，参与细胞增殖与凋亡的调节，相关作用机制仍需深入研究。我们前期在果蝇中筛选到一个新的Hippo通路调节miRNA：miR-285。发现miR-285下调Hippo通路多个下游基因表达，促进细胞凋亡，导致发育异常。本项目旨在此基础上深入研究miR-285通过Hippo通路调节细胞增殖与凋亡的作用机制及其对发育的生理意义。拟通过鉴定miR-285靶基因，明确miR-285调节Hippo通路的分子机制；利用CRISPR/Cas9等技术构建miR-285突变果蝇，检测其细胞增殖、凋亡及发育的异常，探讨发育中miR-285的生理功能，为深入理解Hippo通路的调控网络提供理论依据。

Hippo信号通路最早在果蝇中被鉴定发现，在调节组织器官大小及组织稳态中发挥重要作用。血脑屏障是一种在进化上高度保守的生理性屏障，只选择性的允许一些分子从血液或者体液进入脑组织，防止病原体及一些有害的外源物质进入大脑，从而起到保护大脑和维持正常神经活动进行的作用，然而目前对其在生物体发育过程中形成及维持机制的研究还非常有限。本研究发现了由miR-285-Yki/Mask构成的双重负反馈通路调控Hippo信号通路活性，调节SPG细胞内复制，维护血脑屏障的新机制。我们发现，miR-285在果蝇脑组织中高表达。miR-285通过直接靶向Mask，抑制Hippo通路下游核心转录辅因子Yki的转录活性，下调细胞内复制进程关键因子Cyclin E的表达。miR-285缺失导致SPG细胞中Yki活性的升高以及Cyclin E的异常积累，导致SPG细胞染色体内复制的异常，引起血脑屏障结构缺陷。另一方面，miR-285的表达受到Yki的抑制，过表达yki导致miR-285表达水平下降，因此它们之间构成了双重的负反馈调控。这个负反馈调控通路在调节Yki活性和Cyclin E表达水平，保持SPG细胞内复制正常进行，维护血脑屏障功能中发挥重要作用。此外，过表达miR-285会诱导依赖于Hippo通路的细胞凋亡，该凋亡不依赖于p53或JNK通路。本研究揭示了microRNA以及Hippo信号通路在血脑屏障发育过程中重要的调控作用和机制，为认识血脑屏障生理过程中的调控作用提供了新的理论依据。