Skp2与Ku70相互作用在宫颈癌细胞增殖中的作用研究

It is urgent to illuminate he mechanisms of cervical cancer cell proliferation and to find effective treatment methods. S-phase kinase-associated protein 2 (Skp2) relates to the development and malignant degree of cervical cancer highly, and it is a hot spot for cervical cancer therapy target. However, the exact role of Skp2 in the development of cervical cancer and its regulatory mechanism remain unclear. The results of our mining analysis with the Oncomine database showed that the mRNA levels of both Skp2 and Ku70 increased hand-in-hand in cervical cancer tissues, with a high positive correlation constant (R=0.8), suggesting that the Skp2 and Ku70 might interact with each other in the pathological development of cervical cancer. Further research results proved that the Skp2 and Ku70 proteins bound to each other in cervical cancer cell line HeLa, and Ku70 could help to maintain the stability of intracellular Skp2 and exertion of its biological functions. Thus, Ku70 might promote the deubiquitination of Skp2 by biding it to maintain the stability of Skp2 proteins, finally involved in the development of cervical cancer. The results of the pre-experiments were in accordance with the expectation. In this proposal, we will adopt three cervical cancer cell lines, take the interaction of Skp2 and Ku70 as a research entry point, and apply the in vitro deubiquitination system, in vitro cell lines, and in vivo tumor bearing nude mice models, to carry out further studies of interaction between Skp2 and Ku70, in the hope of discover new mechanisms of Skp2 involved in the development of cervical cancer, and to provide a basis for the development of Skp2 targeted drugs, and new ideas for the prevention and treatment of cervical cancer.

阐明宫颈癌细胞增殖机理，寻找有效治疗手段，是目前亟待解决的问题。S期激酶相关蛋白2（Skp2）与宫颈癌密切相关，是宫颈癌治疗靶标。我们对Oncomine数据库的挖掘分析，发现Skp2与Ku70的mRNA表达水平在宫颈癌组织中均显著升高，并呈正相关性（R=0.8），提示宫颈癌疾病进程中，Skp2与Ku70可能有相互作用。进一步研究发现Ku70可与Skp2相互结合，维持Skp2蛋白的稳定及生物学功能。推测Ku70可能通过与Skp2结合来促使其去泛素化，稳定Skp2蛋白，参与宫颈癌的发生发展。预实验结果与预期相符。本项目拟采用三种宫颈癌细胞系，以Skp2与Ku70相互作用为研究切入点，在体外去泛素化体系、细胞水平与荷瘤裸鼠模型，深入研究Skp2与Ku70的互作的结构与功能关系，阐明Ku70调控Skp2去泛素化的机制，及其参与宫颈癌细胞增殖的机制，为研发Skp2靶向药物及宫颈癌防治提供新思路。

Skp2与多种肿瘤关系密切，通过多种方式参与肿瘤的发生发展，是肿瘤病理机制中的关键蛋白。Skp2的表达水平在宫颈癌组织中也显著升高，且与宫颈癌进展和恶化程度密切相关.本项目的研究基于Skp2与Ku70的相互作用开展，发现Skp2与Ku70在宫颈癌细胞内有直接的相互作用，Skp2可通过与Ku70相互作用调控Ku70的泛素化水平，且Skp2与Ku70的相互作用可调控宫颈癌细胞的增殖、周期与凋亡，凋亡调控机制与Bax信号通路相关。Skp2与Ku70在宫颈癌细胞内的相互作用，且对宫颈癌细胞生物学功能的调控，不受HPV感染背景的影响。我们还定位了Skp2与Ku70的结合位点，根据这个结合位点多肽构建的变异型Ku70表达载体，在细胞水平可抑制宫颈癌、肺癌等相关肿瘤细胞增殖，有希望作为药物治疗潜在靶点进行进一步研究。为深入研究Skp2对宫颈癌细胞增殖的调控作用，我们采用泛素化蛋白组学筛选泛素化水平可受Skp2调控的底物蛋白，我们拟进一步验证这些潜在的底物蛋白，以揭示Skp2参与宫颈癌发生发展的新的病理机制。