RNA去甲基化酶Fto通过下调Ngn3的m6A修饰参与调控胰岛发育的机制研究

The development of pancreatic islet, an endocrine tissue that plays vital role in maintaining gluco-homeostasis, has attracted attention of the science community for decades. As a marker of the endocrine progenitor cells, transcription factor Ngn3 reportedly regulated islets development through multiple downstream pathways, while the mechanism by which modulates Ngn3 expression during embryonic development is not yet known. With bioinformatics analysis and experimental confirmation, our current study showed that Ngn3 transcript contains m6A modifications and is a potential target of Fto, a known RNA m6A demethylase. We further profiled the pancreatic Fto expression during embryonic development and results suggested Fto is negatively related with the Ngn3 expression. Moreover, knockdown of Fto in Min6 cells significantly increased Ngn3 expression. Therefore, we hypothesis that Fto might play important role in regulating Ngn3 m6A modification and its expression, that also possessed undiscovered functions in regulating islets development. In the present study, we design a series of in vivo and in vitro assays to figure out the underline mechanism of Fto in regulating Ngn3 expression. Using the Fto endocrine progenitor cell specific over-expressing mice, we will further explore the undiscovered function of Fto in modulating islets development. Taken together, our research will discover new regulatory mechanism of islets development and reveal an Fto-Ngn3 m6A modification pathway. Hopefully, our work will bring theoretical support of effective islets culture in vitro and benefit the treatment of Type 2 Diabetes in the future.

胰岛对血糖稳态的维持具有重要作用，调控其发育的分子机制研究一直备受关注。已有研究显示，胰岛祖细胞标志物Ngn3直接调控胰岛发育，但机体如何调控Ngn3的表达依旧有很多未知。结合生物信息学分析与实验验证我们发现，Ngn3的转录本存在m6A修饰，且该修饰可被去甲基化酶Fto调控。进一步研究发现，胰岛中Fto与Ngn3的表达水平呈负相关，在胰岛细胞中敲低Fto能够显著上调Ngn3的表达。由此我们推测，在胰岛发育过程中，Ngn3的表达可能受到m6A修饰的调控，而去甲基化酶Fto则介导了这一过程。本项目拟在细胞和动物水平上，借助分子生物学方法和定点敲入小鼠的技术手段，研究Fto调控Ngn3 m6A修饰和表达的分子机制，并探索其对胰岛发育的影响。本项目的开展将有助于明确胰岛发育的调控机制，揭示新的Fto-Ngn3-胰岛发育通路，为体外获得大量有功能的胰岛、改善2型糖尿病血糖稳态带来新的希望。

胰岛对血糖稳态的维持具有重要作用，调控其发育的分子机制研究一直备受关注。Ngn3是调控胰岛发育的核心转录因子，但机体如何调控Ngn3的表达依旧有很多未知。前期研究发现：Ngn3的转录本存在m6A修饰，且该修饰可被去甲基化酶Fto调控；胰岛Fto可显著负调控Ngn3的蛋白表达。据此提出假设：在胰岛发育过程中，Ngn3的表达可能受到m6A修饰的调控，而去甲基化酶Fto则介导了这一过程。本项目在前期研究的基础上，构建了胰岛内分泌祖细胞特异性的Fto敲入鼠，借助多种甲基化测序技术，明确Fto可调控胰岛β细胞的Ngn3 m6A修饰，从而抑制其蛋白表达；进一步，从体内和体外实验两个方面，借助高糖钳夹及胰岛灌流实验，我们发现Fto敲入鼠胰岛功能衰退，血糖清除能力降低。我们的研究进一步明确了Ngn3这一调控胰岛发育的关键转录因子的上游调控机制，为相关疾病的治疗提供了新的潜在靶点。