缺氧环境中DLL4对局部晚期宫颈癌放疗敏感性的影响及其分子机制的研究

Locally advanced cervical cancer (LACC) is one group of cervical cancer, which recurrence rate is high and the prognosis is poor. Because the tumor diameter≥4cm, radiation therapy is the preferred program for patients with LACC. However the radiosensitivity is poor because of the hypoxia due to the large tumor. Therefore, there is an urgent need to find the target gene which can predict and improve the radiosensitivity of LACC. Delta like ligand 4 (DLL4), one of the five notch signaling ligands in mammals, has been recently documented in various malignancies and proven to have an important function in the proliferation, invasion, metastasis, progression, and angiogenesis of malignancies. Our previous findings have revealed that the expression level of DLL4 was upregulated in the LACC tissues, and the DLL4 overexpression was clearly correlated with its radiosensitivity and the expression of HIF-1α and VEGF. Previous studies showed that NF-κB and HIF-1α was involved in angiogenesis of tumor under the hypoxia, and VEGF is a positive regulator factor of DLL4, DLL4 is a negative reguletor factor of VEGF. Therefore, we hypothesize that in the hypoxic environment of LACC, DLL4 promote the development and maturation of the new vessels under the interaction of NF-κB/HIF-1α/VEGF signaling pathway and DLL4/Nothch signaling pathway in order to improve the radiosensitivity. In.the project, we plan to study the effect and mechanism the effect and mechanism of DLL4 gene on the radiosensitivity in hypoxia of locally advanced cervical cancer through three levels of clinical patients-cell line-animal model. Based on this study, the conclusion will provide a theoretical and experimental basis for clinic to predict the prognosis and improve the curative effect for clinical radiotherapy and provide new molecular targets for individualization radiation.

同步放化疗是局部晚期宫颈癌（LACC）首选的治疗手段，但瘤体较大导致的细胞缺氧易引起放疗抵抗。δ样受体4（DLL4）是Notch的重要配体，促进恶性肿瘤中新生血管的成熟和血管网的分化。前期研究中，我们发现LACC中DLL4的表达与放疗敏感性正相关，与HIF-1α和VEGF表达负相关。但DLL4影响肿瘤放疗敏感性的机制，迄今国内外尚无报道。既往文献表明VEGF正向调控DLL4，DLL4负向调控VEGF。由此我们推测：在LACC的缺氧环境中，DLL4通过NF-κB/HIF-1α/VEGF与DLL4/Nothch通路的交互作用，促进血管的正常化，改善细胞缺氧进而发挥放疗增敏作用。为验证该假说，我们拟采用RNA干扰、荧光素酶报告系统及小动物活体成像等技术，明确DLL4在预测和提高LACC放疗敏感性中的作用及机制。本课题将从全新视角阐明DLL4放疗增敏的分子机制，并为LACC的个体化治疗提供新靶点。

背景：宫颈癌是全球女性常见的恶性肿瘤之一，世界范围内，其发病率和死亡率居于女性恶性肿瘤的第四位，在我国，宫颈癌的发病率逐年提升，且呈现年轻化的趋势，仍是严重威胁我国女性健康的主要疾病之一。放射治疗既是中晚期宫颈癌的首选治疗方法，而原发或继发放疗抵抗是限制疗效的主要障碍。因此，深入研究宫颈癌放疗抵抗的分子机制，寻找可以预测宫颈癌预后及放疗敏感性的分子标志物以及发挥放疗增敏作用的治疗靶点，具有一定的理论意义和实用价值。.主要研究内容：Delta样配体 4（Delta-like ligand 4，DLL4）在血管发育和稳态的维持中发挥重要作用，同时在恶性肿瘤中，参与调控新生血管的成熟和血管网的分化，相关研究表明DLL4的表达水平与某些肿瘤的预后密切相关。但是，迄今为止暂无DLL4与宫颈癌放疗抵抗方面的相关报道。因此本研究主要着眼于：①研究DLL4的表达水平与接受同步放化疗的宫颈癌患者的放疗敏感性及预后关系；②应用RNA干扰技术沉默DLL4表达对宫颈细胞增殖能力、细胞周期、凋亡水平和侵袭转移能力的影响；③应用RNA干扰技术沉默DLL4表达对宫颈细胞放疗敏感性的影响，并初步探讨DLL4在宫颈癌放疗抵抗中的机制。.结果：（1） DLL4主要表达于宫颈癌细胞的细胞浆中，其在正常宫颈组织中的表达率明显低于FIGOⅢ期宫颈鳞癌组织。在FIGOⅢ期宫颈癌中，DLL4蛋白的表达水平与临床分期、组织学分级、盆腔淋巴结转移、放疗敏感性和放疗后复发密切相关（P<0.05）。多因素Logistic回归分析结果表明DLL4表达水平是FIGOⅢ期宫颈鳞癌患者是否出现放疗抵抗的独立预测因子之一；（2）抑制DLL4的表达，可以明显降低宫颈癌细胞的迁移和侵袭能力，诱导宫颈癌细胞的凋亡，增强宫颈癌细胞的放疗敏感性，然而对宫颈癌细胞的增殖能力无明显影响。DLL4沉默发挥放疗增敏作用的主要机制可能为：①上调P53表达，促进细胞凋亡；②延迟细胞照射后的DNA损伤修复；③抑制EMT过程和基质金属蛋白酶的分泌。.科学意义：本研究为临床上寻找有效抑制宫颈癌侵袭转移和增加放疗敏感性的分子靶点奠定理论基础。DLL4有望成为下一个有效的宫颈癌放疗增敏的分子靶向治疗的靶点。