信号通路XBP1-p21在细胞周期调控中的分子机制研究

The cyclin-dependent kinase inhibitor p21 is essential in the regulation of cell cycle progression. p21 can inhibit the activity of cyclin-CDK1 and/or -CDK in response to DNA damage stress. p21 mediates G1 phase cell cycle arrest and leads the cells to enter G0 phase，in which cell proliferation and cell cycle are stopped，and the damages are fixed. p21 is also involved in the regulation of apoptosis pathway，a further way to protect the cells when the damages could not be repaired. Thus，p21 is an critical factor for protecting the cells and the whole body by preventing the damaged and/or mutated cells to proliferate. Furthermore，recent reports showed that p21 is also involved in cells senescence and tumor formation. In response to a variety of stresses, tumor suppressor gene p53 and oncogene c-MYC play an important role in regulation of p21 activation. However，the whole mechanism of p21 regulation remains to be elucidated. In an effort to elucidate the mechanism of p21 regulation，in our preliminary experiment，we performed a systematical screening by using shRNA (short hairpin RNA) expression library against transcriptional factors. We discovered a novel p21 regulator，X-box Binding Protein 1 (XBP1). We found that inhibition of XBP1，a factor involved in Unfolded Protein Response (UPR)，significantly upregulated the transcriptional activity of p21, and increased the expression of endogenous p21 protein. XBP1 expression is high in tumor cells，and had been reported to be involved in tumor cell proliferation. However，the detailed mechanism remains to be elucidated. In this project, we aim to reveal the detailed molecular mechanism of XBP1-p21 pathway in cell cycle regulation. We will perform in vitro and in vivo investigations from the following three aspects：(1) Investigate the role of UPR/XBP1 pathway in activating p21；(2) Elucidate the crosstalk regulation of XBP1-p21 pathway with p53- and c-MYC pathway；(3) Investigate the effect of XBP1-p21 pathway on the cell phenotypes and tumor growth. The detailed analysis would not only reveal the role of XBP1 in the p21 regulation mechanism and cell cycle，but also would provide a basis for developing therapeutic strategy regarding diseases related to cell proliferation and/or apoptosis.

细胞周期调控因子p21是一个重要的细胞周期蛋白依赖性激酶抑制剂。当细胞损伤时，p21即能阻止变异细胞增殖，诱发细胞凋亡，从而保护机体组织。既往研究表明p21与细胞DNA修复、凋亡以及肿瘤增殖有密切的联系。尽管已发现p53和c-MYC是p21的重要调控因子，但对p21调控的分子机制尚未完全理清。应用shRNA表达文库对p21转录活性进行筛选，我们发现非折叠蛋白应激因子XBP1是一个新的p21转录调控因子。本课题将从以下三方面展开深入研究：(1)解析XBP1对p21的调控机制和细胞功能的影响; (2)阐明XBP1-p21通路与p53、c-MYC信号分子之间的相互作用; (3)考察XBP1-p21通路对肿瘤生长的影响。通过本项研究拟揭示XBP1-p21信号通路的分子调控机制，以加深对细胞功能调控的理解，为与细胞增殖及凋亡相关的疾病治疗提供新的理论依据。

细胞周期是调控细胞增殖的关键因素，它的紊乱与肿瘤等各种疾病发生密切相关。细胞周期调控是一个严格有序的过程，在这一过程中各种cyclin和cyclin dependent kinase（Cdk）因子起到关键的调控作用。细胞周期因子p21是第一个被发现的Cdk抑制因子，它通过各种Cdk因子调控细胞周期。此外，p21在DNA修复、老化、分化等重要生理现象中也发挥关键作用。然而，目前调控p21的分子机制仍尚未完全被阐明。. 本项目针对细胞周期因子p21的分子调控机制展开了详细的解析。首先，利用p21启动子报告基因和shRNA表达质粒文库针对调控p21转录活性的因子进行大规模基因筛选，从筛选结果中发现非折叠蛋白应激因子X-box Binding Protein 1（XBP1）作为新的p21负调控因子，然后再利用Crispr/Cas9技术构建XBP1缺陷型结肠癌细胞株（HCT116XBP1null），进一步从mRNA和蛋白质层面确认XBP1和p21的负相关性。XBP1被认为是非折叠蛋白应激（UPR）的关键因子，UPR能诱导XBP1的非剪切型XBP1（XBP1-u）剪切成在UPR应激中发挥作用的剪切型XBP1（XBP1-s）。我们发现，当特异性地过表达XBP1-u和XBP1-s时，影响p21转录活性的是XBP1-s前躯体XBP1-u，而不是XBP1-s。我们进一步研究阐明XBP1-u是通过其碳末端结合HDM2/p53信号通路中的HDM2，并阻止HDM2泛素化，从而稳定HDM2蛋白质，促进p53的泛素化，导致p53蛋白质降解，最终负调控p21的转录活性。临床肿瘤组织样品分析也表明，XBP1-u和HDM2在临床结肠癌患者肿瘤组织中呈高表达，而p21的表达明显下降。. 综上所述，本项目发现了XBP1-u在非UPR的条件下对细胞周期因子p21和细胞周期调控的新功能，详细解析了XBP1-u对经典信号通路HDM2/p53的分子调控机制，项目研究揭示了XBP1-u通过HDM2/p53信号通路发挥其独特的重要生理调控意义。由于HDM2/p53通路在细胞周期调控、细胞凋亡、肿瘤形成、老化等基本生物功能方面发挥重要作用，本项目的发现表明XBP1-u极有可能参与到其它重要生理和病理的调控，因此具有重要的科学意义。