泛素连接酶STUB1对宫颈癌放射敏感性的影响及机制研究

Our preliminary results showed that the expression of hTERT was increased in the cytoplasm of the established radioresistant C33AR cervical cancer cells, while the expressions of STUB1 and hTERT in the nucleus were decreased. Moreover, we demonstrated that STUB1 overexpression in C33AR cells increased their radiosensitivity in radiotherapy through induction of telomere dysfunction. Our results suggested that STUB1 could be an important determinant of cellular radiosensitivity in cervical cancer. To confirm this hypothesis, this project aims to study the relationship between the STUB1 and radiosensitivity by measuring the expression of STUB1 in different cervical cancer cell lines with different radiosensitivities, and then investigate the effect of STUB1 on the cancer radiosensitivity by the model with overexpression and silence of gene STUB1 in human cervical cancer cells. We will explore the mechanism of STUB1 in regulating telomere homeostasis and radiosensitivity. Meanwhile, we will use animal tumor models, tumor specimens library data and follow-up data analysis to further study the effect of STUB1 on the radiotherapy in vivo. These findings will enrich the theory of radiosensitivity regulation and provide a potent radiosnsitization target in clinical radiotherapy.

本课题组前期研究发现在宫颈癌放射抗拒细胞模型C33AR中，端粒酶亚单位hTERT蛋白胞浆表达增高、胞核表达降低，同时泛素连接酶STUB1表达降低；进一步研究发现过表达STUB1能降低端粒酶活性、缩短端粒长度、增加C33AR细胞放射敏感性。我们的研究提示STUB1可能是调控宫颈癌放射敏感性的重要靶点。本项目拟进一步探索STUB1在不同放射敏感性的宫颈癌细胞模型中的表达差异，并建立过表达或基因沉默STUB1的稳定转染株，研究STUB1对放射敏感性的影响；阐明STUB1调控端粒稳态的可能机制；揭示STUB1通过端粒稳态调控宫颈癌细胞放射敏感性的可能机制；体内实验验证STUB1对宫颈癌细胞的放射增敏效应；最后对宫颈癌标本库及随访数据进行分析，明确STUB1与放疗疗效的关系，为临床放疗增敏提供新的理论基础。

放射治疗是宫颈癌的主要治疗手段之一，研究宫颈癌细胞内在放射敏感性调节的分子机制对提高宫颈癌的局控率及生存率具有重要的理论和临床意义。前期本课题组在探索端粒酶亚单位hTERT调控放射敏感性的机制过程中，发现hTERT泛素化修饰在端粒稳态维持方面有一定作用，进一步推测与hTERT相互作用的泛素连接酶STUB1可能参与放射敏感性的调控。因此，本课题进一步探索STUB1在不同放射敏感性的宫颈癌细胞模型中的表达差异，并建立过表达或基因沉默STUB1的稳定转染株，研究STUB1对放射敏感性的影响；阐明STUB1调控端粒稳态的可能机制；揭示STUB1通过端粒稳态调控宫颈癌细胞放射敏感性的可能机制；患者标本中检测并分析STUB1表达水平与宫颈癌的关系。结果发现STUB1低表达与宫颈癌不良预后有关；过表达STUB1可通过促进hTERT蛋白胞浆泛素化修饰使hTERT蛋白胞浆储备减少，并通过下调PI3K及AKT，使PI3K/AKT介导的hTERT磷酸化激活减少，从而导致进入胞核有活性的hTERT减少、降低端粒酶活性、缩短端粒、下调大部分端粒相关蛋白，使端粒相对欠稳定，从而使ATM/ATR-Chk1/Chk2-CDC25C介导的细胞周期G2/M期阻滞缩短、Bax/Bcl2比例提高而致细胞凋亡增加，进而增加细胞DNA损伤灶，最终导致放射增敏。这些结果提示过表达STUB1可使宫颈癌细胞放射增敏，为未来进一步在宫颈癌患者肿瘤组织类器官培养中验证过表达STUB1的放射增敏效应并逐步转化至临床提供理论依据及实验基础。