膜蛋白ASTL介导宫颈癌辐射抵抗及其分子机制研究

The incidence rate of cervical cancer is second after breast cancer. Radiotherapy is adopted as a commonly used therapy for patients with cervical cancer. Whereas, mounting clinical and epidemiological studies have reported that patients with cervical cancer exhibit remarkably radioresistance, resulting in poor prognosis. However, the underlying mechanism remains largely unknown. Recent studies have shown that the membrane protein ASTL (Astacin-like metalloendopeptidase) is closely related to the growth of uterine tumors, and can be used as a potential target for the treatment of cervical cancer. Moreover, ASTL is expressed in different tumor tissues and cervical cancer tissues. Our preliminary observation revealed that the combination of ASTL antibody and γ-ray irradiation (IR) could significantly decrease the proliferation of cervical cancer cells, indicating that ASTL plays an important role in regulating the radiosensitivity of cervical cancer cells in vitro. In addition, our results found that radiation caused the translocation of ASTL from membrane to cytoplasm and increased the protein level of ASTL in cervical cancer cells. Furthermore, ASTL up-regulated the expression level and phosphorylated level of AKT, indicating that ASTL participated in the activation of AKT signaling pathway. These results support our hypothesis that ASTL is an important mediator in radiosensitivity of cervical cancer. This object is to investigate the potential mechanism by which ASTL induces the radioresistance of cervical cancer cells through acting as a novel radiation-responding protein by using in vitro and in vivo models. And we will investigate the key role of AKT during the regulation of cervical cancer radiosensitivity. Our finding will provide new insights into the mechanism in regulation of the radiosensitivity of cervical cancer and also provide ASTL as a novel target for individual therapy of cervical cancer patients.

放射治疗是宫颈癌的主要治疗手段之一，但宫颈癌病人产生的耐放射治疗是放疗在宫颈癌治疗中受到限制的主要原因。膜蛋白ASTL与子宫癌细胞的生长及增殖密切相关。我们预实验研究发现通过采用ASTL抗体抑制ASTL蛋白的生物功能，可以显著增强宫颈癌细胞的放射敏感性。辐照可以上调ASTL的表达水平并促进ASTL蛋白从胞膜向胞浆转移。而且，我们还发现ASTL在宫颈癌细胞内可以显著激活蛋白激酶AKT信号通路进而导致肿瘤细胞耐辐射。本项目将在大量预实验研究的基础上，采用宫颈癌体外细胞模型及体内动物模型，深入研究ASTL作为新的辐射应答基因在宫颈癌放射敏感性调节方面的作用和初步机制。期望通过一系列临床前研究，为ASTL作为新的靶向基因在提高临床宫颈癌放射治疗疗效中的应用提供研究依据和实验基础。因此，本研究无论是在宫颈癌放射治疗的基础研究领域还是在今后临床研究上的应用都具有重要的意义和研究价值。

宫颈癌是恶性肿瘤的一种，在妇科恶性肿瘤中排在首位。近年来，我国宫颈癌临床发病呈年轻化趋势，早期宫颈癌大多选择手术治疗与放射治疗，可是当肿瘤体积过大或发展到中晚期的宫颈癌患者很难实施手术，治疗并不理想，且肿瘤容易再次复发甚至发生转移。因此探究宫颈癌细胞的耐辐射机制，进而设计并应用有效的靶向药物配合临床上放射治疗，可以有效控制肿瘤体积，降低宫颈癌的分期，为根治性手术提供保证。膜蛋白Astacin-like metalloendopeptidase (ASTL)，是一个卵母细胞表面相关的锌基质金属蛋白酶，该基因含有431个氨基酸，编码46kDa的蛋白产物。ASTL只在卵巢、子宫癌以及宫颈癌等组织中存在蛋白表达，在其它正常组织中几乎没有表达，同时ASTL的表达水平与子宫癌、卵巢癌等癌症的发生发展密切相关并可以作为对子宫癌治疗的潜在靶点。本项目采用体外细胞和体内动物模型深入研究了ASTL作为新的辐射应答基因在宫颈癌放射敏感性调节方面的作用和分子机制。结果显示，ASTL抗体能够增强宫颈癌细胞的放射敏感性，且MTT实验结果显示ASTL抗体对宫颈癌细胞ME-180的增敏作用会随时间变化逐渐增强。接下来我们选择另外一种宫颈癌细胞HeLa作为研究模型，运用克隆形成、MTT以及EdU等实验方法，进一步证实ASTL的抗体处理或转染含有ASTL干扰序列的过表达质粒均可以增强HeLa细胞的放射敏感性。另外，我们进一步证实了当过表达ASTL蛋白到HeLa细胞后，细胞的放射敏感性显著降低。免疫荧光结果显示辐照能够诱导ASTL由胞膜向胞质转移，同时上调ASTL的蛋白表达水平；同时，实验结果显示ASTL在胞内发挥功能后会重新定位到细胞膜上。Western blot结果显示，ASTL能够在细胞质中发挥功能，进而上调蛋白激酶AKT的表达水平从而激活AKT信号通路。以此为主线，我们通过质谱分析以及转录组学结合芯片分析的方法，筛选出与ASTL相互作用的新型癌蛋白CLPTM1L。最终，我们通过一系列功能实验证实ASTL通过结合CLPTM1L上调AKT表达水平进而调控宫颈癌细胞放射敏感性。本研究对提高临床宫颈癌放射治疗疗效和预后评估的基础研究以及临床应用研究都具有重要的价值和意义。