探討FOXA2啟動子甲基化與入侵性食道鱗狀細胞癌轉移的相關性

Association between FOXA2 promoter methylation and metastasis in invasive esophageal squamous cell carcinoma

吳睿庠(Ruei-Siang Wu)

指導教授 ： 李瑞年

高雄醫學大學/生命科學院/生物醫學暨環境生物學研究所/碩士(2014年)

摘要

食道鱗狀細胞癌為台灣男性癌症十大死因第六位，由於癌細胞容易轉移，五年內的預後存活率低於10%。根據文獻指出，FOXA2可以調控增加細胞轉移抑制因子E-cadherin的表現量進而抑制癌細胞的轉移，但在食道癌中FOXA2調控機制尚不明瞭。首先初步使用MethPrimer software analysis發現FOXA2啟動子存在高量的CpG位點，因此我們認為FOXA2在癌細胞中的低表現量是與啟動子高度甲基化有關係。接著我們使用甲基化特異性聚合酶連鎖反應（methylation-specific polymerase chain reaction, MSP）分析已轉移食道癌檢體及未轉移食道癌檢體中FOXA2啟動子甲基化情形，結果顯示已轉移食道癌檢體中有較高的FOXA2啟動子甲基化現象。根據此結果，我們選擇具有不同轉移能力的食道癌細胞株，確認FOXA2表現與癌症轉移之間的關聯性。首先將食道癌細胞株處理去甲基化藥劑5-Aza-2’-deoxycitidine （5-aza-dC），我們發現FOXA2及E-cadherin表現量的回復，更進一步也觀察到食道癌細胞株的細胞間黏附性增加以及轉移能力下降。接著建立一個包含FOXA2結合位的E-cadherin啟動子序列冷光載體，確認E-cadherin啟動子轉錄活性可由FOXA2所調控，我們發現處理5-aza-dC後，食道癌細胞株中冷光載體上的E-cadherin啟動子轉錄活性上升。另一方面，我們同時觀察到食道癌細胞株經由5-aza-dC處理後再轉染FOXA2 siRNA，E-cadherin的表現量有下降的情形。因此在食道癌細胞中，FOXA2的表現確實能增加E-cadherin表現量進而抑制癌細胞轉移，在未來對於食道癌轉移的研究具有很大的潛力。

關鍵字

FOXA2 ； DNA甲基化 ； 癌症轉移

並列摘要

Esophageal squamous cell carcinoma (ESCC) is the sixth leading cause of cancer death among men in Taiwan. Because of high level of metastasis, the five-year survival rate is below 10%. Forkhead box A2 (FOXA2, also known as HNF3β), can directly upregulate the metastatic suppressor gene E-cadherin, and inhibit cell migration in many kinds of cancer. The mechanism of metastasis regulation in ESCC is yet unknown. Preliminarily, many CpG sites distribute in FOXA2 promoter with MethPrimer software analysis. We considered low FOXA2 expression owing to hypermethylation in cancer cells. Methylation level of FOXA2 was analyzed by methylation-specific polymerase chain reaction (MSP) in metastatic and non-metastatic ESCC samples. Higher methylation level of FOXA2 promoter in metastatic ESCC was discovered. Therefore, we selected different ESCC cell lines based on metastasis ability to confirm the relationship between FOXA2 expression and metastasis. First, 5-Aza-2’-deoxycitidine (5-aza-dC) was used to demethylate FOXA2 promoter in ESCC cell lines. FOXA2 and E-cadherin reexpression were confirmed by western blot. Furthermore, low cell migration level was confirmed by wound healing assay and Boyden chamber assay after treating with 5-aza-dC. In the meantime, the stronger cell-cell adhesion was discovered. Based on the above results, we constructed E-cadherin promoter luciferase reporter system to confirm FOXA2 influence on E-cadherin promoter. The induction of promoter activity was observed after treating with 5-aza-dC in ESCC cell lines. On the other hand, reexpression of FOXA2 by 5-aza-dC in ESCC cell lines was inhibited by FOXA2 siRNA and E-cadherin expression was also inhibited. According to our results, reexpression of FOXA2 can upregulate E-cadherin expression and has the potential to inhibit metastasis in esophageal cancer.

並列關鍵字

FOXA2 ； DNA methylation ； metastasis

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