XAB2下调通过抑制POLR2A表达促进细胞衰老的分子机制研究

Cellular senescence is closely related to geriatric diseases, so it is of great significance to find the key molecules in regulating cellular senescence. XAB2 plays critical roles in gene expression regulation and DNA damage repair, but little is known about the function and mechanism of XAB2 in cellular senescence. In previous work, we have reported that XAB2 deficiency led to cell cycle arrest (Cell Death Dis, 2016). Our preliminary data show that down-regulation of XAB2 inhibits the expression of RNA polymerase II subunit POLR2A, knockdown of XAB2 and POLR2A both increases SA-β-Gal activity. In addition, both XAB2 and POLR2A genes are downregulated in H2O2-induced senescent cell models. Thus we will first verify the regulation of XAB2 on cellular senescence via POLR2A through senescent cell and animal models, using knockdown, overexpression and rescue experiments combined with phenotype analysis. Next we will identify key domains and interacting proteins of XAB2 involved in regulating POLR2A expression. We will also investigate the molecular mechanism of XAB2 in regulating POLR2A expression on transcription, pre-mRNA splicing, RNA degradation and stability, protein degradation and stability, respectively. Finally we will explore the effect of XAB2 or POLR2A on p53/p21 and p16/pRB signaling pathways. The project aims to elucidate the mechanism on the regulation of cellular senescence by XAB2, which may eventually lead to potential target in the prevention and treatment of aging and geriatric diseases.

细胞衰老与老年病密切相关，寻找调控细胞衰老的关键分子意义重大。XAB2参与基因表达调控与DNA损伤修复，但在细胞衰老中的作用与机制尚未揭示。申请人曾报道XAB2下调阻滞细胞周期（Cell Death Dis，2016）。预实验发现下调XAB2抑制RNA聚合酶II亚基POLR2A表达，二者敲低后均增强SA-β-Gal活性，且皆在H2O2诱导衰老细胞模型中低表达。据此，本项目拟首先通过衰老细胞与动物模型，采用敲低、过表达与恢复实验结合表型分析进一步明确XAB2通过POLR2A影响细胞衰老，进而鉴定调控POLR2A表达的XAB2关键结构域及相互作用蛋白，并分别从转录、剪接、RNA及蛋白的降解与稳定性等方面验证XAB2调控POLR2A表达的分子机制，最后探讨XAB2和POLR2A对p53/p21与p16/pRB信号通路的影响。本项目将阐明XAB2调控细胞衰老的机制，为衰老及老年病防治提供新的靶点。

细胞衰老与老年病密切相关，寻找调控细胞衰老的关键分子意义重大。XAB2参与基因表达调控与DNA损伤修复，但在细胞衰老中的作用与机制尚未揭示。在基金委的支持下，本项目旨在研究XAB2调控细胞衰老的作用与机制，为衰老及老年病防治提供新的靶点。首先，建立了复制型衰老和H2O2诱导的衰老细胞模型，结果显示XAB2在衰老细胞中的蛋白表达明显下调，在皮肤成纤维细胞系HFF1中敲低XAB2，结果显示XAB2下调后SA-β-GAL活性明显增强，G2/M期明显上升，细胞增殖被明显抑制，表明XAB2下调会促进细胞衰老。其次，敲低XAB2会造成POLR2A的mRNA与蛋白水平皆明显下调，POLR2A在复制型衰老和H2O2诱导的衰老细胞中的蛋白表达明显下调，POLR2A敲低后同样会增强SA-β-GAL活性，敲低XAB2后过表达POLR2A则能够恢复相应衰老表型，表明XAB2下调通过抑制POLR2A的表达促进细胞衰老。再次，利用双荧光素酶报告系统检测实验、ChIP实验、新生RNA分离鉴定实验、RNA-seq、体外剪接实验、TMT标记的蛋白质组学实验、mRNA稳定性实验等证实，XAB2敲低导致的POLR2A下调并不依赖于转录，而主要在RNA剪接和稳定性水平调控。另外，利用蛋白免疫沉淀结合质谱分析鉴定与XAB2相互作用的蛋白，GO分析显示与XAB2相互作用的蛋白主要参与剪接，敲低SNRNP200、EFTUD2、SNW1、PRPF8、PLRG1及AQR导致POLR2A RNA与蛋白皆明显下调，XAB2截短实验证实XAB2的2-4及11-12号TPR结构域对POLR2A的表达至关重要，IP实验进一步表明XAB2通过该结构域与SNW1相互作用从而维持POLR2A的表达。最后，探讨了XAB2和POLR2A与p53/p21衰老信号通路的关系，结果显示在HFF1中敲低XAB2或POLR2A，p53与p21表达显著提高，而敲低XAB2后过表达POLR2A则能够部分下调p53与p21的表达，表明XAB2通过POLR2A对细胞衰老的调控受到p53/p21通路的介导。综上，本项目阐明了XAB2调控细胞衰老的作用与机制，为衰老及老年病防治提供了新的线索。