MT1-MMP诱导口腔癌细胞上皮间质转化并促其侵袭转移的机制研究
本文关键词:MT1-MMP诱导口腔癌细胞上皮间质转化并促其侵袭转移的机制研究,由笔耕文化传播整理发布。
侵袭和转移是恶性肿瘤特征之一,也是口腔癌患者致死的主要原因。越来越多的研究表明,上皮间质转化(epithelial-to-mesenchymal transition, EMT)与恶性肿瘤的发展密切相关。膜型基质金属蛋白酶-1(Membrane Type1MatrixMetalloproteinase, MT1-MMP)是一种以活性形式表达在细胞表面上的蛋白酶,被认为是影响细胞周围微环境的因子之一,参与降解细胞外基质组成成分从而促进肿瘤侵袭和细胞迁移。目的:探讨MT1-MMP在诱导口腔癌细胞EMT及促其侵袭转移过程中的作用机制。方法:首先,构建pEGFP-N1-MT1-MMP真核表达质粒;通过细胞转染的方法,获得稳定表达细胞株SCC9-N(对照组)及SCC9-M(实验组);通过实时定量PCR、免疫蛋白印迹技术、免疫荧光显微方法检测上皮和间质标志物的表达改变,观察细胞的形态学变化;应用粘附实验、侵袭实验、划痕实验检测细胞生物学特性的改变。其次,通过细胞流式仪、MTT及单克隆形成实验检测发生EMT之后的细胞是否获得肿瘤干细胞(cancer stem cells, CSCs)样的特性。最后,通过慢病毒干扰载体转染细胞,下调口腔癌细胞中MT1-MMP的表达水平,进一步检测MT1-MMP对口腔癌细胞侵袭能力的影响。结果:成功构建pEGFP-N1-MT1-MMP真核表达质粒;过表达MT1-MMP诱导SCC9发生了明显的EMT,细胞呈细长的成纤维细胞样形态,上皮标志物E-cadherin,, cytokeratin18和β-catenin表达下降,而间质标志物vimentin和fibronectin的表达明显上升;MT1-MMP所诱导的细胞形态改变增加了转录抑制因子Twist和ZEB的表达并且是通过抑制E-cadherin的转录,使细胞粘附能力减弱而侵袭能力增强。MT1-MMP不仅诱导SCC9细胞发生了EMT,同时使其获得了CSCs样的特性,增殖能力减弱,但能够形成新的单克隆仍具有自我更新能力,CSCs表面标记物CD24表达降低,对化疗药物具有一定的抗性和抗凋亡特性。通过慢病毒干扰载体转染细胞,下调SCC25中MT1-MMP的表达后导致MMP-2、MMP-9和MMP-13表达均下降;同时,过表达MT1-MMP的SCC9细胞中MMP-2,MMP-9和MMP-13的表达均上升。以上结果说明口腔癌细胞中MT1-MMP可调控MMP-2,MMP-9和MMP-13的表达。另外,下调MT1-MMP使侵袭能力较强的SCC25细胞侵袭能力减弱;相反的,过表达MT1-MMP使SCC9细胞获得了较强的侵袭能力,说明MT1-MMP可增强口腔癌细胞侵袭能力。结论:本实验证实过表达MT1-MMP诱导口腔癌细胞发生EMT进而促其侵袭转移,并且使其获得CSCs样的特性。MT1-MMP通过调控MMP-2,MMP-9和MMP-13的表达从而影响口腔癌细胞的侵袭能力。这些有关MT1-MMP分子功能的相关研究,有可能为口腔癌的临床治疗提供新的靶点。
Tissue invasion and metastasis are acquired abilities of cancer and related to the deathin oral squamous cell carcinoma (OSCC). Emerging observations indicate that theepithelial-to-mesenchymal transition (EMT) is associated with tumor progression.Membrane Type1Matrix Metalloproteinase (MT1-MMP) is a cell surface proteinasein an active form, which is known as one of the factors that influence the pericellularmicroenvironment. MT1-MMP is involved in degrading extracellular matrixcomponents that can promote tumor invasion and cell migration.Objective: In this study, we intend to investigate the role of MT1-MMP in inducingEMT and promoting tumor cell invasion and metastasis in OSCC.Methods: First, the eukaryotic expression vector pEGFP-N1-MT1-MMP wasconstructed. We utilized SCC9cells stably transfected with an empty vector(SCC9-N) or a vector encoding human MT1-MMP (SCC9-M) to study the role ofMT1-MMP in EMT development. Real-time RT-PCR, western blotting,immunofluorescence microscopy were used to detect the changes of the epithelial andmesenchymal markers. Adhesion, invasion and wound-healing assay were performedto measure the biological properties of the cells. Next, the CSC-like characteristics inSCC9-M cells were evaluated by flow cytometry, MTT, colony-forming assay.Furthermore, we utilized SCC25cells transfected with a vector with a scrambledmiRNA sequences (SCC25-mock) as experiment control or the most effectivelentivirus-miRNA interference vector (SCC25-miRNA-M) to downregulation ofMT1-MMP. Real-Time RT-PCR, western blotting, transwell invasion assay were performed to further determine the role of MT1-MMP in oral cancer cell invasion.Results: First, the eukaryotic expression vector pEGFP-N1-MT1-MMP wasconstructed. Upon up-regulation of MT1-MMP, SCC9-M cells underwent EMT, inwhich they presented a fibroblast-like phenotype and had a decreased expression ofepithelial markers (E-cadherin, cytokeratin18and β-catenin) and an increasedexpression of mesenchymal markers (vimentin and fibronectin). We furtherdemonstrated that MT1-MMP-induced morphologic changes increased the level ofTwist and ZEB, and were dependent on repressing the transcription of E-cadherin.These activities resulted in low adhesive, high invasive abilities of the SCC9-M cells.Next, MT1-MMP-induced transformed cells exhibited cancer stem cell (CSC)-likecharacteristics, such as low proliferation, self-renewal ability, resistance tochemotherapeutic drugs and apoptosis, and expression of CSCs surface markers.Furthermore, upon downregulation of MT1-MMP in SCC25cells caused a decreasedlevel of MMP-2, MMP-9and MMP-13. Meanwhile, up-regulation of MT1-MMP inSCC9cells could also activate the expression of MMP-2, MMP-9and MMP-13. Thisresult revealed that oral cancer cell MT1-MMP expression was associated with theexpression of MMP-2, MMP-9and MMP-13. Downregulation of MT1-MMP inSCC25cells caused the more aggressive cancer cells decreased the invasive ability.By contrast, the less aggressive SCC9cells obtained high invasive ability byoverexpression of MT1-MMP. This data demonstrated that cancer cell MT1-MMPexpression affected the invasive ability of cancer cells.Conclusions: Our study indicates that overexpression of MT1-MMP induces an EMTand promotes cancer cell invasion and metastasis in OSCC. This phenotypetransformation results in the acquisition of CSC-like properties in SCC9cells. Oralcancer cell MT1-MMP is correlated with the expression of MMP-2, MMP-9andMMP-13, affects cancer cell invasion, and leads to a remodeling of the tumor microenvironment. These aspects of MT1-MMP function in cancer invasion andmetastasis are giving our approaches to a better understanding of OSCC therapy.
MT1-MMP诱导口腔癌细胞上皮间质转化并促其侵袭转移的机制研究 中文摘要6-8Abstract8-10前言11-131. 材料和方法13-292. 结果29-483. 讨论48-524. 结论52-53参考文献53-58文献综述58-72 参考文献65-72攻读学位期间发表文章等情况72-73致谢73
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本文关键词:MT1-MMP诱导口腔癌细胞上皮间质转化并促其侵袭转移的机制研究,由笔耕文化传播整理发布。
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