Lin28a调控ES细胞naïve-primed状态转变机制研究

Lin28a plays a key role in organ development and pluripotency maintenance of embryonic stem (ES) cells. With the development and application of single cell technology, researches demonstrated that the expression of Lin28a is heterogenetic in ES cells. ES cells population with higher expression level of Lin28a gradually deviate from the naïve ES cells population and show mesoderm-like characters. Our preliminary data showed that Lin28a expression was increased in primed state ES cells compared with that in naïve state. The upregulation of Lin28a was observed at early stage of ES cells differentiation followed by downregulation at late stage. Ectopic expression of Lin28a in ES cells was sufficient to promote primed features, including flattened colony morphology, downregulation of naïve related genes, and upregulation of primed pluripotency markers. Notably, we firstly uncovered that Lin28a knockdown significantly increased Dppa3 (specific expressed in naïve state ES cells, but absent in epiblast stem cells and primed ES cells) expression and consequently elevated the proportion of Dppa3 positive ES cells. . In this proposal, we hypothesize that Lin28a might be as a gatekeeper in ES cells pluripotency transition and early committed cell lineages. Based on this hypothesis, we will investigate the mechanisms of Lin28a in ES cells pluripotent state transition and early differentiation. Firstly, we will explore the roles of Dppa3 during the naïve-primed state conversion regulated by Lin28a. And then we will focus on let-7, signaling pathway, protein translation and DNA methylation mechanisms to examine other mechanisms of Lin28a during this process. We are hopeful that this project would uncover new functions of Lin28a in ES cells pluripotency maintenance and reveal the mechanisms during this process. It will help to understand the mechanisms involved in pluripotency conversion and lineage commitment further laying a foundation for clinical application of human ES cells.

Lin28a作为发育及多能性相关基因备受关注，最新报道及我们的研究显示Lin28a高表达促使ES细胞向primed状态转变，Lin28a下降显著提升naïve状态调控因子Dppa3表达，提示其在多能状态转变中具有重要作用。基于此提出科研假设：Lin28a调控naïve-primed状态转变及早期分化，该作用可能通过调控Dppa3实现。从该科研假设出发，首先进一步验证Lin28a在ES细胞多能状态转变及早期分化中的作用；进而从两方面探讨机制：一是针对前期发现的Lin28a负向调控Dppa3表达，探究Dppa3在Lin28a调控多能状态转变中的作用及机制；二是从let-7、蛋白翻译及DNA甲基化等Lin28a作用模式入手积极探讨这些机制在多能状态转变调控中的作用。该课题开展有助于了解ES细胞多能性维持及谱系分化机制，并为人naïve-ES细胞培养体系及定向分化体系建立提供借鉴意义。

Lin28a是一种具有保守结构域的RNA结合蛋白，在胚胎干细胞多能性维持、肿瘤发生、代谢及发育过程中起重要作用。在胚胎干细胞（ES细胞）中，Lin28a 一直作为一个多能性调控因子备受关注，但其在Naïve-Primed状态转变中的作用及机制不清。为了解 Lin28a 在 ES 细胞多能性维持中的作用，尤其是在Naïve - Primed 状态转变中的作用我们开展了本课题研究。通过研究发现Lin28a在 primed状态ES细胞中高表达，在naïve状态下表达降低；进一步利用过表达及敲低等方法发现Lin28a在ES细胞naïve-primed状态转变调控中发挥重要作用，其表达升高促使ES细胞向primed状态转变，反之ES细胞向naïve状态转变；进一步我们详细探讨了Lin28a在naïve-primed状态转变调控中的作用机制，发现Lin28a调控ES细胞多能状态转变部分是通过负向调控Dppa3实现的。Lin28a敲低后显著性促进Dppa3的表达，使Dppa3阳性细胞群体比例升高。通过营救实验发现Dppa3能够逆转由Lin28a所引起的ES细胞多能性状态的转换。随后我们又对Lin28a调控Dppa3的机制进行了探讨，发现Lin28a通过抑制let-7的成熟，升高let-7的靶基因DNA甲基转移酶（Dnmt3a，Dnmt3b）表达，使Dppa3启动子区域甲基化，Dppa3启动子区域的高甲基化导致Dppa3表达下降，进而调控ES细胞由Naïve State向Primed State进行转换。利用let-7模拟物（let-7 mimic）对let-7进行活性恢复实验时，发现let-7 mimic可降低Dnmt3a/b表达，Dppa3甲基化水平下降，Dppa3表达升高，以维持ES细胞处于Naïve 状态。综上所述，我们的发现Lin28a可以通过Dppa3调控ES细胞naïve-primed状态转变，Lin28a对Dppa3的调控作用是通过let-7/Dnmt3a/b介导的。此外，我们还研究了Stat3信号通路在ES细胞多能性状态转变中的作用，发现Stat3激活在ES细胞Naïve向Primed状态转换中发挥了促进作用。