内质网应激诱导ERp29及其靶向蛋白促进结直肠癌转移的分子机制研究

Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide. Metastasis is the main cause compromising patients with colorectal cancer. Endoplasmic reticulum (ER) and endoplasmic reticulum stress (ERS) play an important role in tumor progression. ER is widely existed in eukaryotes and it has the function of both calcium storage and protein folding. Accumulation of unfolded or misfolded protein in the ER can lead to ER stress. To adapt to the occurrence of ERS, cells activate highly conserved stress response, called the unfolded protein reaction (UPR). ER stress and UPR have been implicated in diabetes, tumors, neurodegenerative diseases and cerebral ischemia. In the previous study, we have made an attempt to identify proteins associated with colorectal cancer metastasis by comparing the metastatic potentials among different clone spheres and their paternal cell line SW480 through two-dimensional electrophoresis (2-DE) separation and mass spectrometry (MS). We fortunately have found out that Endoplasmic reticulum protein 29 (ERp29) was closely correlated with metastasis of colorectal cancer. ERp29 as an essential endoplasmic reticulum factor, how did ERp29 promote CRC genesis and metastasis through endoplasmic reticulum stress? To review literatures, we have not found any researches were involved in the molecular mechanism of ERp29 in CRC. So, we try to establish a model of ERS in vitro, and observe how the ERp29 changes under ERS. As all know, protein-protein interaction networks at the whole level lead to tumorigenesis and progression. Therefore, for further investigating molecular mechanism of ERS in CRC genesis and progression, high throughput proteomic techniques, including 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) and FLAG Pull-down, were used to screen ERp29-related signal molecules and interacting proteins. It may be significant to explicit the mechanism of CRC progression and metastasis after screening ERp29-modulated proteins and further investigating their functions. The aim of the present study was to observe the effects of ERp29 on the regulation of a variety of important intercellular proteins under ERS, which could contribute to canceri-genesis and progression of CRC, and to propose the underlying mechanism of these molecular interactions. Further validation studies would provide clues for elucidate molecular mechanism of CRC genesis and progression.

结直肠癌转移是影响患者治疗效果和导致患者死亡的主要原因，内质网应激在肿瘤发生发展过程中起着关键性作用。本项目前期发现内质网应激相关蛋白ERp29与结直肠癌的生长与侵袭行为有关。ERp29在肿瘤诱导的内质网应激中发挥作用，但其作用方式以及作用在哪条信号通路上目前还不清楚。本研究模拟在体内质网应激诱导状态，以ERp29为研究对象，通过双向荧光差异凝胶电泳技术筛选ERp29相关信号通路的关键分子，采用FLAG Pull-down 技术筛选ERp29靶向作用蛋白，重点探讨内质网激诱导下ERp29及其靶向蛋白在结直肠癌增殖、侵袭及转移的作用机制，阐述内质网应激诱发结直肠癌转移的相关信号通路，为深入研究结直肠癌的发生、发展及转移机制提供新的思路。

结直肠癌是全球范围内高危害的恶性肿瘤之一。在中国，结直肠癌的发病率在逐年激增。侵袭和转移是结直肠癌患者后期复发和死亡的重要原因。因此，寻找到与结直肠癌侵袭和转移相关的预测指标，阐明结直肠癌转移发生发展的分子机制是目前结直肠癌研究的重要内容。近年有研究表示，内质网应激可参与肿瘤的发生发展过程。人体内许多因素可以引起内质网稳态失衡。使得内质网内正常的蛋白质合成过程受到干扰，导致内质网内未发生折叠或者有错误折叠的蛋白质增多，诱发内质网应激。同时细胞会通过一系列的信号转导途径对上述反应做出应答，被称作非折叠蛋白反应（UPR），主要表现为增强蛋白质的翻译折叠能力，停滞新的蛋白的加工合成，加快错误折叠蛋白的降解以及增加分子伴侣蛋白的表达。目前研究已经发现的内质网分子伴侣蛋白有GRP98、GRP74、ERp29等。其中ERp29蛋白是一种新型的内质网蛋白，表达于人体多种组织中。分子量为29KD,定位于12号染色体上。前期临床标本研究表明ERp29与结直肠癌患者的不良预后有关。但是ERp29是否在结直肠癌诱导的内质网应激中起作用，以及它的作用方式目前还不清楚。. 本项目将通过衣霉素体外刺激结直肠癌细胞株模拟内质网应激，探讨内质网应激是否诱导ERp29及ERp29与其下游相关分子蛋白在结直肠癌的增殖，侵袭和转移中的作用机制研究。1.成功构建了结直肠癌细胞株（SW620和HCT116）中的内质网应激模型，并阻断了内质网应激效应。2.内质网应激过程中结直肠癌细胞株的体外增殖和侵袭能力降低。3.结直肠癌细胞株内质网应激过程中ERp29蛋白表达增高。 4.ERp29过表达可以促进结直肠癌细胞株的体外克隆形成能力和生长侵袭能力及体内的转移能力。5.利用蛋白质组学技术筛选出ERp29相关蛋白SSR4，初步研究表明结直肠癌细胞株内质网应激与MET过程有关。本研究为结直肠癌的预防和治疗提供新的临床理论依据。