組織因子途徑抑制素第二型在口腔鱗狀細胞癌中 是一個經常受表觀遺傳調控而靜默的抑癌基因

Tissue factor pathway inhibitor-2, a tumor suppressor gene, is frequently silenced by epigenetic regulation in oral squamous cell carcinoma

賴怡卉(Yi-Hui Lai)

指導教授 ： 戴建國

國立中正大學/理學院/生命科學系分子生物研究所/博士(2014年)

摘要

口腔鱗狀細胞癌的早期診斷與良好的預後有關，因此，我們試圖尋找有效的生物標記，做為口腔癌的早期識別以改善口腔癌患者的存活和預後。透過甲基化微陣列晶片分析，我們發現口腔癌檢體中有一基因：組織因子途徑抑制素第二型(tissue factor pathway inhibitor-2, TFPI-2)，是一個具有潛力的臨床預測指標，亦是涉及口腔癌惡性程度的關鍵因子。為了確認TFPI-2在口腔組織檢體中是否具有高度甲基化的現象以及表現量受到抑制的情況，我們利用亞硫酸氫鹽定序法，定量甲基化特異性PCR，和焦磷酸定序法進行評估TFPI-2基因啟動子的甲基化程度，並進一步探討TFPI-2的甲基化高低與口腔癌病患的癌化程度之間的關係。研究結果證實: 在口腔癌組織中TFPI-2甲基化程度高於正常口腔組織，且口腔癌中TFPI-2靜默表現受到DNA甲基化和染色質組蛋白修飾所調控。而在細胞實驗中，我們以5’-aza-2’-deoxycytidine和trichostatin A處理口腔癌細胞，透過定量反轉錄PCR分析確認TFPI-2基因的表達的確透過表觀遺傳調控而靜默。另一方面，我們亦建立TFPI-2過度表達的口腔癌細胞進行細胞增殖、細胞凋亡、癌細胞遷移、侵襲測定，以及癌細胞轉移動物實驗，來探討TFPI-2是否為腫瘤抑制基因。結果發現TFPI-2可藉由抑制基質金屬蛋白酶第二型的活性來降低口腔癌細胞的侵襲性，影響裸鼠體內癌細胞的轉移。綜合以上，我們相信TFPI-2基因在口腔癌細胞中經常會因為過度甲基化而使其表現量降低，顯示TFPI-2在口腔癌癌化過程中扮演著一個重要的抑制角色，避免癌細胞容易轉移而趨向惡性發展。我們期望TFPI-2的甲基化相關研究可以提供口腔癌病人預後的生物指標，並有助於更了解口腔癌發展的分子機制，以期應用於未來治療口腔癌的策略。

關鍵字: 口腔鱗狀細胞癌，DNA 甲基化，組織因子途徑抑制素第二型，基質金屬蛋白酶第二型，癌細胞轉移

關鍵字

組織因子途徑抑制素第二型 ； DNA 甲基化 ； 口腔鱗狀細胞癌 ； 基質金屬蛋白酶第二型 ； 癌細胞轉移

並列摘要

The treatment of oral squamous cell carcinoma (OSCC) following early detection is associated with good outcomes. Therefore, the survival and prognosis of OSCC patients could be hugely improved by identifying reliable biomarkers for the early diagnosis of the disease. Our previous methylation microarray analysis results have suggested that tissue factor pathway inhibitor-2 (TFPI-2) is a potential clinical predictor as well as a key regulator involved in OSCC malignancy. To confirm, methylation of the TFPI-2 promoter in oral tissue specimens was evaluated by bisulfite sequencing assay, quantitative methylation-specific PCR, and pyrosequencing assay. The significance of the differential pattern of TFPI-2 hypermethylation between normal oral and OSCC tissues makes it a promising biomarker for the early detection of OSCC. TFPI-2 expression can be reactivated after combined treatment with 5’-aza-2’-deoxycytidine (5-azaDC) and trichostatin A (TSA), revealing a synergistic regulation of DNA methylation and histone deacetylation in TFPI-2 silencing. We also investigated whether TFPI-2 plays a role as a tumor suppressor by establishing TFPI-2-overexpressing OSCC cells and subjecting them to in vitro cellular proliferation, migration, and invasion assays, as well as an in vivo metastasis assay. Restoration of TFPI-2 counteracted the invasiveness of OSCC by inhibiting the enzymatic activity of matrix metalloproteinase-2. Our data suggest strongly that TFPI-2 is a down-regulated tumor suppressor gene in OSCC, probably involving epigenetic silencing mechanisms. The loss of TFPI-2 expression is a key event for oral tumorigenesis, especially in the process of tumor metastasis.

Keywords: oral squamous cell carcinoma, DNA methylation, TFPI-2, matrix metalloproteinase-2, metastasis

並列關鍵字

tissue factor pathway inhibitor-2 ； DNA methylation ； oral squamous cell carcinoma ； matrix metalloproteinase-2 ； metastasis

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