诱导癌细胞发生上皮间质化(EMT)的新分子:MT2-MMP及其作用机制研究

Epithelial-mesenchymal transition (EMT) is an important initial phase for the invasion and metastasis of cancer cell, however, its mechanism is unclear. Multiple members of the matrix metalloproteinase (MMP)gene family are expressed during tissue remoldeling events associated with grwth, development and neoplastic events. Current efforts in the field remain focused on attempts to identify the most critical participants among the 28 known MMPs. However, new studies suggest that two of the four type 1 transmembrane MMPs in this family, i.e.,MT1-MMP and MT2-MMP, play singular roles in terms of proteolyzing the extracellular matrix, controlling cell invasion and regulating cell growth as well as differentiation. While most attention remains focused on MT1-MMP, with many years of MT1-MMP research and pre-experiment on MT2-MMP, we found that: ① MT2-MMP (ie, MMP15) was highly expressed in a variety of cancer tissues; ② MT2-MMP expression was negatively correlated with the E-cadherin expression in cancer cell lines; ③ non-invasive cancer cell line HCT116 stably transfected with MT2-MMP changed from epithelioid to spindle-like morphology; ④ E-cadherin expression was downregulated in the MT2-MMP stably transfected cells. All the above new findings suggest that MT2-MMP may induce cancer EMT; ⑤ By sequence alignment, binding sites for NF-κB and snail1, respectively, were found in the promoter region of the MT2 gene, suggesting that EMT inducer TGF-β1 may regulate MT2-MMP expression through activating NF-κB, and, Wnt1 through activating snail1, resulting in cancer cell EMT and metastasis. Multiple levels including animal models, clinical cancer tissue specimens, cytology and molecular biology in our research will be utilized to elucidate the role and molecular mechanism of MT2-MMP in inducing cancer EMT, whereby providing potential and noval target molecules for anticancer drug research.This study has important scientific significance and potential application value.

上皮间质化（EMT）是癌细胞浸润转移的重要启动阶段，目前机制不清。我们在多年MT1-MMP的工作基础上预实验发现：① MT2-MMP(即MMP15)在多种癌组织中高表达；②在肿瘤细胞系中与E-钙粘蛋白呈负相关表达模式；③首次发现稳转MT2-MMP后非侵袭性癌细胞系HCT116由上皮样转变为纺锤样形态；④ E-钙粘蛋白表达下调。以上发现提示MT2-MMP可能是诱导癌EMT的新的分子。⑤ 序列比对发现MT2-MMP启动子区分别有NF-κB、snail1的结合位点，推测EMT诱导因素TGF-β1可能通过激活NF-κB调控MT2-MMP表达，Wnt1通过激活snail1调控MT2-MMP的表达，导致EMT和侵袭转移。拟从动物模型、临床癌组织标本以及细胞学、分子生物学等多个层次阐明MT2-MMP诱导EMT的作用及分子机制，为有针对性地研发抗癌药物提供靶分子，本研究有重要的科学意义和潜在应用价值。

上皮间质化（EMT）是癌细胞浸润转移的重要启动阶段，目前机制不清。本课题自申请下来以后即开始按照计划开展工作，在四年时间里组织课题组成员按照研究计划开展各项研究内容，基本阐明了MT2-MMP导致肿瘤发生EMT的分子机制。通过分子生物学，免疫组化，细胞生物学等技术方法，研究了MT2-MMP与E-钙粘蛋白在癌组织标本中的分布特点，发现与肺癌、乳腺癌、结肠癌等成病理相关性，MT2-MMP表达使细胞形态发生转变，由上皮样向纤维样细胞转变，说明发生了EMT， MT2-MMP分子能降解E-钙粘蛋白及细胞连接蛋白ZO-1，从而使细胞缺失典型的上皮样形态，并发生EMT，MT2-MMP影响了Vimentin及FN1等的表达，说明从上游调控的途径影响这些纤维样细胞的标志分子的表达，对MT2-MMP降解作用起关键作用的结构域及活性位点是蛋白分子中260谷氨酸位点，当突变为丙氨酸则这些作用消失，MT2-MMP受到TGF-β1的调控的调控从而增强EMT，MT2-MMP与肿瘤细胞侵袭转移的关系得到明确，MT2-MMP能增强肿瘤细胞侵袭转移，而其突变体没有该作用。培养研究生四名，发表SCI收录文章两篇。