细胞复制性衰老过程中mTORC1靶基因的筛选与功能研究

Cell replicative senescence is fundamental to organismal aging, which correlates to aging-related diseases, such as neurodegenerative disease and cancer. During replicative senescence, many genes alter their expression levels, and their gene products also contribute to the initiation or development of cell senescence. However, few studies focus on the screening research on that process; and which mRNA’s translation is regulated by replicative senescence is largely unknown. This study is based on that mTORC1 plays an essential role in replicative senescence. By using polysomal profiling and RNA-seq, we try to screen novel senescence-associated genes whose translation is regulated by mTORC1 during cell replicative senescence. Furthermore, we attempt to analyze those genes’ function on both cellular level and organismal level by utilizing lentivirus-mediated RNA knockdown in cells and CRISPR/Cas9-mediated gene knockout in mice, separately. This study will enable us to have a deeper understanding of aging process and aging-related disease. Screening of novel senescence-associated genes will help to find new targets for the treatment of aging-related diseases and provide important guidance for therapies.

细胞复制性衰老是个体衰老的基础，与衰老相关疾病，如神经退行性疾病和肿瘤等的发生和发展紧密相连。在细胞复制性衰老的过程中，许多基因的表达水平会发生改变，而这些细胞衰老相关基因的表达产物也会影响细胞衰老进程；但目前对此过程的系统性研究较少，尤其是在基因表达调控的mRNA翻译层次更是未见报道。本项目拟以mTORC1在细胞复制性衰老过程中发挥重要功能为突破口，采用多聚核糖体纯化技术和RNA高通量测序技术来筛选在细胞复制性衰老过程中其翻译水平受到mTORC1调控的mRNA种类，寻找复制性衰老相关基因。进一步利用慢病毒介导的RNA干涉技术和CRISPR/Cas9敲除小鼠技术分别在细胞层次和个体层次研究筛选出的基因在衰老过程中的作用。本项目研究结果将加深我们对于衰老过程和衰老相关疾病的认识；本项目筛选出的衰老相关基因，有利于我们寻找治疗衰老相关疾病的靶点，为研发新的治疗方法提供重要参考和依据。

细胞衰老是个体衰老的基础，深入研究细胞衰老的调控机制，找到阻止细胞衰老的因子，对于延缓个体衰老具有重要意义。目前大规模筛选调节细胞衰老的因子主要集中在转录调控水平，而在翻译水平进行筛选罕见报道。本项目利用多聚核糖体纯化联合RNA测序分析，在成纤维细胞衰老过程中筛选翻译水平发生变化的mRNA种类，有助于发现新的衰老相关基因。我们构建了人和小鼠复制性细胞衰老模型，检测mTORC1的活性在衰老过程中的变化模式，发现细胞复制性衰老过程中mTORC1的活性下降。由于mTORC1调控含有5’ TOP（末端寡聚嘧啶结构）结构的mRNA的翻译过程，因此我们重点关注翻译水平随衰老过程下调的mRNA种类，成功筛选出小鼠复制性细胞衰老模型中的两个衰老相关基因C1和RpsX，并确认其在细胞衰老过程中发挥作用。此外，我们建立了新型基于生物素标记的多聚核糖体纯化体系，该系统能够摆脱对大型设备的依赖，使得基因翻译水平的检测更加简便和有效。在本项目的资助下，我们还取得了下列成果：（1）筛选出一个新的端粒相关蛋白SBDS，并揭示其调节端粒长度的机制，成果发表在Cell Reports杂志；（2）发现维生素C具有提高间充质干细胞组织修复功能的作用，成果发表在Stem Cell Research & Therapy上；（3）发明一种大量获得抗衰老蛋白Klotho的方法，申请专利一项。其他成果正在撰写论文中，争取早日发表在高水平杂志上。