E3泛素连接酶CDC20调控FoxO1促进DNA损伤修复影响膀胱癌放疗敏感性的作用及机制研究

Radiation resistance is the main reason affecting the efficacy of radiotherapy for bladder cancer, however, the specific regulation mechanism is still unknown. E3 ubiquitin ligase CDC20 may be an important factor that mediates radiation resistance. Our preliminary studies found that E3 ubiquitin ligase CDC20 regulated multiple biological activities including cell cycle, proliferation, invasion and migration of cancer cells. Pilot experiment results showed that CDC20 knockdown significantly increased the radiosensitivity of bladder cancer cells and promoted accumulation of FoxO1. Therefore, we speculate that E3 ubiquitin ligase CDC20 could ubiquitinate FoxO1 and promote DNA damage repair, leading to radiation resistance in bladder cancer. In order to test this hypothesis，this project will detect the expression of CDC20 in clinical specimens of bladder cancer with different radiation sensitivity. Next, we will investigate the effect of CDC20 and FoxO1 on DNA damage repair and radiosensitivity of bladder cancer by apoptosis assay, clonogenic assay and tumor xenograft model. Finally, we will reveal molecular mechanism how CDC20 affects DNA damage repair and radiotherapy sensitivity by regulating FoxO1. This study is significant for understanding the mechanism of radiation resistance, and will provide us with new theoretical and experimental basis for seeking new strategies to intervene radiation resistance of bladder cancer .

辐射抵抗是影响膀胱癌放疗疗效的主要原因，但具体调控机制不明。既往研究发现E3泛素连接酶CDC20介导肿瘤辐射抵抗，我们已有的研究表明： CDC20参与调控肿瘤细胞的周期、增殖、转移和侵袭；预初实验结果显示沉默CDC20可增加膀胱癌细胞内FoxO1蛋白积累，抑制DNA损伤修复，增加放疗敏感性。因此，我们推测E3泛素连接酶CDC20可泛素化降解FoxO1，促进DNA损伤修复，从而引起膀胱癌辐射抵抗。为验证该假说，我们将检测膀胱癌临床标本中CDC20表达并分析其与膀胱癌放疗敏感性的相关性；其次，使用细胞凋亡、克隆形成和荷瘤裸鼠等实验明确CDC20和FoxO1对膀胱癌DNA损伤修复和放疗敏感性的调控作用；最后，阐明CDC20通过调控FoxO1促进DNA损伤修复诱导膀胱癌辐射抵抗的机制。本项目的实施将阐明膀胱癌辐射抵抗新机制，为寻求干预膀胱癌辐射抵抗新策略提供理论基础和实验依据。

放射治疗是肿瘤患者的常规治疗方法，但患者经常出现远处转移和复发，导致放射治疗预后较差。此外，到目前为止，研究人员尚未探究清楚膀胱癌放疗抵抗的潜在机制。我们在本研究中探究膀胱癌放射耐受的分子机制。首先，我们建立两株辐射抵抗(IR)膀胱癌细胞株，T24R和5637R。我们利用CCK-8法检测细胞活力，创伤愈合法检测细胞迁移能力，Transwell室法检测细胞侵袭能力。此外，通过Western blotting、免疫沉淀和免疫荧光染色探讨Cdc20在调控IR细胞上皮间质转化(epithelial-to-mesenchymal transition, EMT)中发挥的功能。我们的数据显示辐射抵抗细胞呈现EMT特性，并且Cdc20在辐射抵抗细胞中表达显著升高。此外，我们还发现沉默Cdc20部分通过调控FoxO1降解逆转辐射抵抗细胞中EMT表型。总之，我们的研究结果表明Cdc20的失活或FoxO1的激活可能是克服膀胱癌放射耐受的潜在策略。