基質金屬蛋白脢組織抑制因子3在口腔癌致癌過程與表觀遺傳學的研究

中山醫學大學機構典藏 CSMUIR > 口腔醫學院 > 口腔醫學研究所 > 研究計劃 > Item 310902500/10303

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/10303

Title:	基質金屬蛋白脢組織抑制因子3在口腔癌致癌過程與表觀遺傳學的研究 Study of Tissue Inhibitor of Metalloproteinase 3 Expression and Epigenetic Regulation in Oral Cancer
Authors:	林巧雯
Contributors:	口腔科學研究所
Keywords:	牙醫學;生物技術(醫) 口腔癌;基質金屬蛋白酶組織抑制因子3;癌症轉移;甲基化 oral cancer;TIMP3;metastasis;methylation
Date:	2014
Issue Date:	2015-02-25T09:17:33Z (UTC)
Abstract:	口腔癌(oral cancer)是台灣地區發生率和死亡率增加速度最快的癌症。因此。口腔癌的防治為台灣重要議題之一。基質金屬蛋白水解酶 (matrix metalloproteinases; MMPs)及其抑制劑，基質金屬蛋白酶組織抑制因子(Tissue inhibitors of metalloproteinases; TIMPs)在口腔癌的致癌及轉移過程中扮演很重要的角色。因此，本實驗室一直致力於參與細胞外基質(extracellular matrix; ECM)分解的相關基因如MMPs及其抑制劑TIMPs與口腔相關疾病的研究，尤其是在口腔癌的致病機轉。 TIMPs是MMPs的內生性抑制劑，能專一性的調控MMPs的活性。MMPs對於腫瘤的侵襲轉移及細胞外基質的降解扮演重要的角色。因此。TIMPs在調控腫瘤增生轉移與維持細胞外基質的平衡方面顯得格外重要。TIMP3為TIMPs的一員，它是TIMPs family中唯一能與ECM結合的分子，主要調節細胞週期與細胞分化。先前的研究指出，TIMP3能抑制黑色素瘤細胞的轉移並促進癌細胞的凋亡。另外，TIMP3可藉由影響VEGF與VEGF受體的結合能力來抑制血管新生。有學者發現在甲狀腺癌細胞株內其TIMP3啟動子區域有高度甲基化的現象，並且與癌症的轉移能力有關。但是TIMP3的表現及其甲基化程度與口腔癌的相關性卻少有文獻探討。本實驗室初步觀察TIMP3在口腔癌病人腫瘤組織和周邊正常組織的表現與其啟動子甲基化的情形。結果發現口腔癌患者的腫瘤組織相較於周邊正常組織，有較低的TIMP3表現量，且甲基化程度較高。因此，這促使我們想要利用口腔癌檢體組織及口腔癌細胞實驗來進一步釐清TIMP3所扮演的角色。本計畫擬於第一年收集更多口腔癌患者的腫瘤組織與周邊正常組織，利用定序方式確認其TIMP3啟動子甲基化的程度，並利用免疫組織染色和ELISA來分析口腔癌患者和對照組其TIMP3表現量的差異，更進一步分析其表現量與口腔癌的轉移與否及一些臨床數據做相關性的分析。目前本實驗室也已建立TIMP3 overexpression的口腔癌細胞株，擬於第二年利用TIMP3 overexpression與knockdown的細胞株觀察口腔癌細胞在不同 TIMP3表現量下，是否會改變細胞轉移與侵襲能力，並探討其訊息傳遞路徑。此外，也利用甲基化轉移酶抑制劑decitabine處理口腔癌細胞株，觀察是否可回復TIMP3的表現與細胞的轉移能力。在第三年的部分，本計畫擬探討TIMP3是否透過Epithelial-mesenchymal transition(EMT) 路徑影響細胞轉移的能力，並利用雞胚胎絨毛膜血管生成試驗等觀察TIMP3在血管生成的調控，最後利用動物實驗觀察TIMP3 overexpression和knockdown口腔癌細胞株對裸鼠腫瘤生長及血管新生所造成的影響。本計畫希望能結合臨床檢體與基礎研究，以釐清TIMP3在口腔癌的致癌與轉移過程的調控與其啟動子甲基化程度對口腔癌生成所造成的影響。 In Taiwan, oral cancer has the highest rate of increasing incidence and mortality of all cancers. Therefore, preventing the occurrence of oral cancer is a critical issue. Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) have crucial roles in the pathogenesis and metastasis of cancer. Our laboratory has extensively studied extracellular matrix-degrading proteases, such as MMPs and TIMPs, and we are particularly interested in the pathogenesis of oral cancer. TIMPs are endogenous MMP inhibitors and exhibit specificity in regulating MMP activity. MMPs play a crucial role in tumor invasion and the metastasis and degradation of extracellular matrix(ECM). Therefore, TIMPs are particularly vital in regulating tumor proliferation and metastasis and maintaining balance of the ECM. TIMP3, a member of the TIMP family, is the only substance that can bind with the ECM and its main role is regulating the cell cycle and cell differentiation. Previous studies have indicated that TIMP3 can inhibit melanoma cell metastasis and induce cancer cell apoptosis. TIMP3 can also inhibit angiogenesis by affecting the binding ability of VEGF and VEGF receptors. Researchers have learned that the promoter region of the TIMP3 gene in thyroid cancer cell lines is highly methylated, and this methylation is correlated with cancer cell invasion ability. However, few studies have focused on the correlation between TIMP3 expression or methylation and oral cancer. Our laboratory has previously investigated the expression and methylation of TIMP3 in the cancer tissue and surrounding normal tissue of oral cancer patients. We learned that in oral cancer patients, cancer tissues expressed lower levels of TIMP3 and higher levels of methylation compared with those of the surrounding normal tissues. This motivated us to further clarify the role of TIMP3 by applying oral cancer tissue samples and cell lines in our experiments. In the first year of this project, we plan to collect additional oral cancer patient-tissue samples (both cancer tissue and surrounding normal tissue) and verify the level of promoter methylation in TIMP3 by conducting DNA sequencing. In addition, we plan to analyze the differences in TIMP3 expression levels in oral cancer patients and control group subjects using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) to further determine whether expression level is correlated with tumor metastasis and to evaluate the correlation of other clinical data. We have completed the construction of a TIMP3 overexpression oral cancer cell line. In the second year of this project, we plan to use these TIMP3 overexpression and knockdown cell lines to investigate whether different levels of TIMP3 expression causes alterations in oral cancer cell metastasis and invasion and further clarify the signal transduction pathway. In addition, we plan to treat the cell lines with the methyltransferase inhibitor decitabine and determine if the TIMP3 expression and cell metastasis abilities are recoverable. In the third year of this project, we will investigate whether TIMP3 affects cell metastasis abilities through epithelial-mesenchymal transition (EMT) and will apply chick embryo chorioallantoic membrane in angiogenesis tests to understand how TIMP3 regulates angiogenesis. Finally, we will conduct animal testing on nude mice to observe the influence of TIMP3 overexpression and knockdown oral cancer cell lines in tumor growth and angiogenesis. In this project, we will combine clinical samples and basic research to clarify the regulation of TIMP3 in oral cancer pathogenesis and metastasis and the effects of TIMP3 promoter methylation of oral cancer.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/10303
Appears in Collections:	[口腔醫學研究所] 研究計劃

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