MUC1对食管鳞癌EMT过程中TGF-β信号通路的作用及调控机制研究

China is a high incidence area of esophageal cancer. Despite recent improvements in treatments, the 5-year survival rate of esophageal cancer continues to hover around 20%. As invasiveness and metastasis are important biological characteristics of malignant neoplasms and also the leading causes of poor prognosis in esophageal cancer patients, it is very important to explore the molecular mechanisms occurring during the invasion and metastasis in esophageal cancer. .Increasing evidences have shown that epithelial-mesenchymal transition (EMT) plays an essential role in tumor progression and metastasis. EMT is a fundamental multistep process not only in development, but also in wound healing, fibrosis and invasion, and metastasis of tumor cells. Without it, multicellular organisms would be incapable of getting past the blastula stage of embryonic development. However, in cancer, the epithelial tumor cells become more invasive after undergoing EMT and access the circulatory system through intravasation, resulting in dissemination of cancer cells to distal loci from the primary tumor. Many signal pathways have contributed to the induction of EMT, such as transforming growth factor-β (TGF-β), Wnt, and Notch. TGF-β signaling has been shown to play an important role in EMT. Several lines of evidences indicate that cancer cells often increase their production of active TGF-β, which not only triggers cells EMT and allows them to become invasive, but also enhances angiogenesis in close proximity to the tumor microenvironment, providing an exit route for migratory mesenchymal cells. .MUC1 is a proteoglycan with extensive O-linked glycosylation of its extracellular domain. Mucins line the apical surface of epithelial cells in the lungs, stomach, intestines, eyes and several other organs. Overexpression of MUC1 is often associated with colon, breast, ovarian, lung and pancreatic cancers. In our previous study, we found that it is helpful to diagnose occult lymph node micrometastasis by detecting the expression of MUC1 mRNA in lymph nodes of pN0 esophageal squamous cell cancer patients. And lymph node micrometastasis may predict poor prognosis of the patients after radical operation. Although MUC1 is associated with the invasive forms of esophageal cancers, the mechanisms of which regulate this malignant behavior remain elusive. Based on previous studies, we boldly presumed that overexpression of MUC1 in esophageal cancer cells triggers the molecular process of EMT, which translates to increased invasiveness and metastasis. .As increased TGF-β signaling was a key effector of EMT in cancer progression and metastasis, in our present study, we purpose to explore the critical signaling molecules correlated of MUC1 in TGF-β signaling pathway and clarity the mechanisms of MUC1 that are responsible for regulating the process of EMT. It would have a great effect on unraveling the metastatic cascade and might lead to a novel intervention for metastatic disease.

上皮细胞-间质细胞转化（EMT）是恶性上皮肿瘤发生发展的基础，但人们对肿瘤细胞中诱导维持EMT状态的信号机制并不十分清楚。TGF-β信号通路是肿瘤转移过程中诱导EMT发生的一个关键通路，TGF-β与经典及非经典Wnt三条信号通路协同作用诱导激活细胞EMT程序，之后以自分泌的方式维持最终的间质细胞状态。我们前期研究发现MUC1基因在食管癌组织及转移淋巴结中存在高表达，与食管癌淋巴结隐匿性微转移、食管癌术后早期复发及不良预后密切相关，最新研究亦发现MUC1能通过诱导癌细胞发生EMT而促其侵袭。本课题拟在前期工作基础上以TGF-β信号通路为切入点，采用RNA干扰技术、高通量组学分析等方法深入研究MUC1基因调控对食管癌EMT转变、侵袭转移等生物学行为的影响，揭示食管癌细胞中控制、维持EMT的相关关键信号分子并阐明其调控机制，为食管癌的基因治疗提供新的靶点和方向。

食管鳞癌是我国常见消化道肿瘤，作为一种高侵袭性恶性肿瘤，病人预后较差，约半数病人于术后2~3年内复发。我们前期研究发现MUC1基因在食管癌组织及转移淋巴结中存在高表达，与食管癌淋巴结隐匿性微转移、食管癌术后早期复发及不良预后密切相关。本研究旨在进一步明确MUC1在食管鳞癌中的表达及其在食管鳞癌侵袭转移过程中的功能，并初步探索其作用机制。本研究通过免疫组化方法进一步检测MUC1在食管鳞癌中的表达并分析其与临床病理及预后的关系。同时通过构建MUC1过表达及shRNA抑制食管鳞癌细胞系分析MUC1对食管鳞癌生物学行为的影响。并分析MUC1在食管鳞癌细胞系EMT过程中发挥的作用。本研究通过免疫组化方法及qRT-PCR检测93例食管鳞癌患者病理组织，并与20例正常食管黏膜上皮组织相比较，发现了MUC1在食管鳞癌中表达上调，并且发现其表达与患者淋巴结转移，术后复发及不良预后呈正相关。我们进一步检测了食管鳞癌细胞系KYSE150，ECA109及TE-1中MUC1mRNA的表达，发现与正常食管上皮细胞系HET-1相比，各食管癌细胞系中MUC1的表达水平均有不同程度的上调。为了进一步研究MUC1在食管鳞癌发生发展过程中的作用，我们构建了MUC1过表达及MUC1siRNA慢病毒载体，成功构建了稳定的MUC1过表达及MUC1抑制Eca109细胞系。体外实验证实MUC1明显促进了Eca109细胞的增殖，迁移及侵袭能力。同时，我们通过western-blot方法检测了E-cadherin，N-cadherin及vimentin等EMT标志物在MUC1过表达及空白对照Eca109细胞系中的表达水平，结果显示MUC1过表达细胞株中E-cadherin的水平较对照组明显减低，而N-cadherin及vimentin的表达水平明显增高，这证实了MUC1在食管鳞癌EMT的过程中发挥着重要作用；但在MUC1抑制Eca109细胞系中并未发现下调MUC1能够逆转EMT过程，EMT的几个分子指标并无显著变化。为了进一步明确MUC1在食管鳞癌细胞中发挥作用的机制，我们利用基因芯片技术对MUC1过表达及MUC1抑制Eca109细胞系进行全基因检测，与空白对照组Eca109细胞系相比较，我们发现MUC1过表达组中TGF-β通路及MAPK通路有显著变化。