本文關(guān)鍵詞：MT1-MMP誘導(dǎo)口腔癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化并促其侵襲轉(zhuǎn)移的機(jī)制研究，由筆耕文化傳播整理發(fā)布。

        侵襲和轉(zhuǎn)移是惡性腫瘤特征之一，也是口腔癌患者致死的主要原因。越來(lái)越多的研究表明，上皮間質(zhì)轉(zhuǎn)化(epithelial-to-mesenchymal transition, EMT)與惡性腫瘤的發(fā)展密切相關(guān)。膜型基質(zhì)金屬蛋白酶-1(Membrane Type1MatrixMetalloproteinase, MT1-MMP)是一種以活性形式表達(dá)在細(xì)胞表面上的蛋白酶，被認(rèn)為是影響細(xì)胞周?chē)h(huán)境的因子之一，參與降解細(xì)胞外基質(zhì)組成成分從而促進(jìn)腫瘤侵襲和細(xì)胞遷移。目的：探討MT1-MMP在誘導(dǎo)口腔癌細(xì)胞EMT及促其侵襲轉(zhuǎn)移過(guò)程中的作用機(jī)制。方法：首先，構(gòu)建pEGFP-N1-MT1-MMP真核表達(dá)質(zhì)粒；通過(guò)細(xì)胞轉(zhuǎn)染的方法，獲得穩(wěn)定表達(dá)細(xì)胞株SCC9-N（對(duì)照組）及SCC9-M（實(shí)驗(yàn)組）；通過(guò)實(shí)時(shí)定量PCR、免疫蛋白印跡技術(shù)、免疫熒光顯微方法檢測(cè)上皮和間質(zhì)標(biāo)志物的表達(dá)改變，觀察細(xì)胞的形態(tài)學(xué)變化；應(yīng)用粘附實(shí)驗(yàn)、侵襲實(shí)驗(yàn)、劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞生物學(xué)特性的改變。其次，通過(guò)細(xì)胞流式儀、MTT及單克隆形成實(shí)驗(yàn)檢測(cè)發(fā)生EMT之后的細(xì)胞是否獲得腫瘤干細(xì)胞(cancer stem cells, CSCs)樣的特性。最后，通過(guò)慢病毒干擾載體轉(zhuǎn)染細(xì)胞，下調(diào)口腔癌細(xì)胞中MT1-MMP的表達(dá)水平，進(jìn)一步檢測(cè)MT1-MMP對(duì)口腔癌細(xì)胞侵襲能力的影響。結(jié)果：成功構(gòu)建pEGFP-N1-MT1-MMP真核表達(dá)質(zhì)粒；過(guò)表達(dá)MT1-MMP誘導(dǎo)SCC9發(fā)生了明顯的EMT，細(xì)胞呈細(xì)長(zhǎng)的成纖維細(xì)胞樣形態(tài)，上皮標(biāo)志物E-cadherin，， cytokeratin18和β-catenin表達(dá)下降，而間質(zhì)標(biāo)志物vimentin和fibronectin的表達(dá)明顯上升；MT1-MMP所誘導(dǎo)的細(xì)胞形態(tài)改變?cè)黾恿宿D(zhuǎn)錄抑制因子Twist和ZEB的表達(dá)并且是通過(guò)抑制E-cadherin的轉(zhuǎn)錄，使細(xì)胞粘附能力減弱而侵襲能力增強(qiáng)。MT1-MMP不僅誘導(dǎo)SCC9細(xì)胞發(fā)生了EMT，同時(shí)使其獲得了CSCs樣的特性，增殖能力減弱，但能夠形成新的單克隆仍具有自我更新能力，CSCs表面標(biāo)記物CD24表達(dá)降低，對(duì)化療藥物具有一定的抗性和抗凋亡特性。通過(guò)慢病毒干擾載體轉(zhuǎn)染細(xì)胞，下調(diào)SCC25中MT1-MMP的表達(dá)后導(dǎo)致MMP-2、MMP-9和MMP-13表達(dá)均下降；同時(shí)，過(guò)表達(dá)MT1-MMP的SCC9細(xì)胞中MMP-2，MMP-9和MMP-13的表達(dá)均上升。以上結(jié)果說(shuō)明口腔癌細(xì)胞中MT1-MMP可調(diào)控MMP-2，MMP-9和MMP-13的表達(dá)。另外，下調(diào)MT1-MMP使侵襲能力較強(qiáng)的SCC25細(xì)胞侵襲能力減弱；相反的，過(guò)表達(dá)MT1-MMP使SCC9細(xì)胞獲得了較強(qiáng)的侵襲能力，說(shuō)明MT1-MMP可增強(qiáng)口腔癌細(xì)胞侵襲能力。結(jié)論：本實(shí)驗(yàn)證實(shí)過(guò)表達(dá)MT1-MMP誘導(dǎo)口腔癌細(xì)胞發(fā)生EMT進(jìn)而促其侵襲轉(zhuǎn)移，并且使其獲得CSCs樣的特性。MT1-MMP通過(guò)調(diào)控MMP-2，MMP-9和MMP-13的表達(dá)從而影響口腔癌細(xì)胞的侵襲能力。這些有關(guān)MT1-MMP分子功能的相關(guān)研究，有可能為口腔癌的臨床治療提供新的靶點(diǎn)。

    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誘導(dǎo)口腔癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化并促其侵襲轉(zhuǎn)移的機(jī)制研究

中文摘要6-8Abstract8-10前言11-131. 材料和方法13-292. 結(jié)果29-483. 討論48-524. 結(jié)論52-53參考文獻(xiàn)53-58文獻(xiàn)綜述58-72    參考文獻(xiàn)65-72攻讀學(xué)位期間發(fā)表文章等情況72-73致謝73

  本文關(guān)鍵詞：MT1-MMP誘導(dǎo)口腔癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化并促其侵襲轉(zhuǎn)移的機(jī)制研究，由筆耕文化傳播整理發(fā)布。