高尔基体磷蛋白3介导的辐射抗性及机制研究

It has been confirmed that several subcellular organelles including the nucleus involve in radiation resistance, an important factor in radiation protection and radiotherapy. However, it is unknown whether there is a definite relationship between the Golgi apparatus/related proteins and radiation resistance. Recently, Golgi phosphoprotein 3 (GOLPH3), a resident Golgi protein, was identified as a linker between DSB and radiation resistance. GOLPH3 was reported to mediate the resistance to DNA-damaging agents via promoting Golgi dispersal. Furthermore, some signaling pathways regulated by GOLPH3 were revealed to protect cells from cell death, potentially related with the radiation resistance. Herein, we hypothesize that GOLPH3 can mediate radiation resistance. And the hypothesis was supported by our previous studies which showed that GOLPH3 was involved in the Golgi dispersion induced by radiation and affected radiation resistance. Further investigation shown below will be performed in the present research: (1) Exploring the pattern of the Golgi dispersal induced by radiation, and clarifying the possible mechanism of Golgi dispersal mediated by GOLPH3. (2) Elucidating the possible mechanism of radiation resistance relying on GOLPH3 in the aspects of the Golgi dispersion and the anti-cell death pathway. We hope these results will promote clarifying the role of the Golgi apparatus and related proteins in radiation resistance, and provide theory and data base for the radiation protection and radiation sensitization by targeting GOLPH3.

辐射抗性是放射性损伤防护和放疗实施的基础和重要依据。核外细胞器在辐射抗性中的作用被逐渐揭示，但高尔基体及其蛋白在辐射抗性中的作用尚未明确。高尔基体磷蛋白3（GOLPH3）作为DNA双链断裂（DSBs）与高尔基体的纽带，介导高尔基弥散，抵抗DNA损伤类药物的“杀伤”；且GOLPH3调控的多条抗死亡信号通路与辐射抗性存在关联，提示GOLPH3具有介导辐射抗性的潜在可能。申请人前期实验结果证实GOLPH3介导辐射后高尔基体弥散，并参与辐射抗性的形成。本项目拟开展进一步研究:（1）探索辐射后高尔基体弥散的规律性，探明GOLPH3介导辐射后高尔基体弥散的机制。（2）从GOLPH3介导的高尔基体弥散以及调控的抗细胞死亡通路两方面，探究GOLPH3参与形成辐射抗性的机制。研究结果将有助于廓清高尔基体及相关蛋白在辐射抗性形成中的作用，为靶向GOLPH3的辐射防护和增敏提供理论指导和实验依据。

肺癌是发病率和死亡率均居于全球第一位的恶性肿瘤。放疗是肺癌治疗的主要方式之一，而放射抗性极大地限制了放疗的疗效。因此探索放射抗性的分子基础，发现新的增敏靶点进而提高放疗敏感性是目前放射生物学研究的热点之一。高尔基体磷蛋白3（Golgi phosphoprotein 3, GOLPH3）是一种新发现的、主要位于高尔基体反面网状结构上的癌蛋白。研究表明，GOLPH3与肿瘤的发生发展、迁移与侵袭、肿瘤细胞干性和化疗抵抗等密切相关。本研究通过TCGA数据库分析和免疫组化技术，证实了GOLPH3在肺腺癌（Lung adenocarcinoma, LUAD）中高表达。本研究通过克隆存活实验和细胞凋亡检测，证明了干扰GOLPH3表达可以提高LUAD的放射敏感性。进一步地通过检测微核形成和γ-H2AX 焦点/蛋白水平，证实了GOLPH3对放射后DSB修复的促进作用，下调GOLPH3表达促进放射后微核的形成、减弱细胞修复DNA双链断裂（DNA double-strand break, DSB）的能力，提高了放射对细胞的杀伤作用。机制研究发现GOLPH3具有维持EGFR蛋白稳定性的作用，通过参与放射后表皮生长因子受体（Epidermal growth factor receptor, EGFR）的入核，调控DNA修复复合体（DNA-PK）活化，进而促进放射后DSB的修复。在裸鼠移植瘤模型中，靶向GOLPH3也呈现出显著的辐射增敏作用。本研究阐明了GOLPH3/EGFR/DNA-PK信号通路对放射后DNA损伤修复的调控机制，同时也为临床放疗提供了一种新的潜在放射增敏分子靶点。