探討FOXA2基因表現與食道鱗狀上皮細胞癌遷移的相關性

Study of the relationship between FOXA2 gene expression and cell migration in esophageal squamous cell carcinoma

李秉蓁(Ping-Chen Lee)

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系碩士班/碩士(2015年)

摘要

食道鱗狀上皮細胞癌是食道癌中最主要的類型，佔總病例的90%，上皮-間質轉化是造成食道癌病患癌細胞轉移的原因之一，其中，上皮-間質轉化的關鍵基因CDH1，是主要調控細胞間黏附的相關因子。根據之前文獻報導指出，在CDH1的啟動子區域有4個FOXA2的轉錄結合位，且FOXA2可以直接上調控CDH1的表現並抑制細胞遷移。由先前的實驗結果證實，經由甲基化抑制劑 (5-aza-dC)處理食道癌細胞株後，FOXA2的表現回升，確實也增加CDH1的表現量，進而抑制癌細胞的遷移，在未來對於食道癌遷移的研究具有很大的潛力。然而，FOXA2的基因表現與食道鱗狀上皮細胞癌遷移的相關性尚未明瞭，因此，本研究為了更進一步探討，分別以三種不同的調控方式進行檢測：以TGFβ1處理使FOXA2基因達到高度甲基化狀態、將FOXA2基因表現默化、將FOXA2蛋白過表現。在未經任何藥物處理前，我們利用甲基化特異性聚合酶連鎖反應 (Methylation-specific polymerase chain reaction, MSP)以及西方點墨法先了解FOXA2及CDH1的甲基化狀態及蛋白表現，結果發現在TE-2、81T、81T-4及146T四株食道癌細胞株，FOXA2基因啟動子均為高度甲基化狀態，CDH1則均為未甲基化狀態；而FOXA2蛋白除了在146T有大量表現外，TE-2、81T及81T-4細胞株均呈低下。有文獻指出，轉化生長因子-β1 (TGFβ1)可以刺激DNA甲基轉移酶 (DNMTs)的活性，並促使基因發生甲基化。146T細胞株經由TGFβ1處理後，FOXA2甲基化程度回升，且FOXA2及CDH1的蛋白表現均下降。此外，在細胞遷移能力方面，146T細胞株經由TGFβ1處理後，在傷口癒合分析實驗 (Wound-healing assay)中，能夠誘導細胞遷移，但在細胞遷移能力分析實驗 (Transwell migration assay)中，遷移能力沒有顯著差異。接著，我們建立一個包含FOXA2轉錄結合位的CDH1啟動子序列冷光載體，證實CDH1啟動子的轉錄活性是由FOXA2所調控，結果顯示，81T、81T-4及146T細胞株經由TGFβ1處理後，81T及81T-4細胞株的CDH1啟動子轉錄活性有劑量依賴性的遞減，但146T細胞株卻沒有差異。此外，81T及81T-4細胞株經由5-aza-dC恢復FOXA2的表現後，能夠被FOXA2-human siRNA抑制其蛋白表現量，且CDH1的蛋白表現也同時被抑制。最後，我們將81T-4細胞株轉染pcDNA3.1-FOXA2使FOXA2蛋白過表現，發現CDH1的表現也跟著回升。根據以上結果證實，在食道癌中，FOXA2的基因表現可藉由DNA甲基化修飾所調控，進而影響CDH1的表現，最後影響癌細胞的遷移能力。期望FOXA2可以成為未來食道癌診斷與治療的生物標靶。

關鍵字

食道癌 ； 基因甲基化 ； 細胞遷移

並列摘要

Esophageal squamous cell carcinoma (ESCC) is the major type of esophageal cancer, it is about 90%. Epithelial-mesenchymal transition (EMT) contributes to cancer cell migration in patients with ESCC. E-cadherin (CDH1), an EMT marker, is one of the genes which regulates the cell-cell adhesion. Previous study indicated that there are four FOXA2 transcription binding sites on CDH1 promoter, and FOXA2 can upregulate CDH1 expression directly and inhibits cell migration. According to the previous results, when the ESCC cell lines were treated with 5-Aza-2’-deoxycytidine (5-aza-dC), which could cause gene demethylation, FOXA2 expression was reexpressed. Upregulation of CDH1 expression increases the potential to cell migration in esophageal cancer. However, the relationship between FOXA2 gene expression and cell migration in ESCC cell lines is still unclear. Therefore, in this study, we want to investigate further mechanism. We detected the FOXA2 expression by three different ways: restore the methylation level by treated with transforming growth factor beta 1 (TGFβ1), silencing the transcription of FOXA2, and overexpression of FOXA2 protein. By methylation-specific polymerase chain reaction (MS-PCR) and Western blot, we had found that FOXA2 was hypermethylated and CDH1 was demethylated. The protein expression of FOXA2 was not expressed in TE-2, 81T and 81T-4 cell lines, but 146T expressed in high amount. In previous studies, TGFβ1 has been reported that would increase the expression of DNA methyltransferase (DNMTs) and caused gene methylation. After treated 146T cell line with TGFβ1, FOXA2 was methylated. The protein expression of FOXA2 and CDH1 were decreased with the addition of TGFβ1. Besides, cell migration of 146T cell line was induced after treated with TGFβ1 by wound-healing assay, but it was not significant different in transwell migration assay. We contructed CDH1 promoter luciferase reporter system to confirm CDH1 promoter activity was influenced by FOXA2. The data showed that after treated ESCC cell lines with TGFβ1, the CDH1 promoter activity was decreased in a dose dependent manner in 81T and 81T-4 cell lines, but 146T was not changed. Besides, the reexpression of FOXA2 which induced by 5-aza-dC, was inhibited by FOXA2-human siRNA and the expression of CDH1 was also inhibited in 81T and 81T-4 cell lines. Finally, we transfected the pcDNA3.1-FOXA2 to overexpress the FOXA2 protein, the expression of CDH1 was also increased in 81T-4 cell lines. Our findings indicate that FOXA2 gene expression could be regulated by DNA methylation, and influence the CDH1 expression, finally cause the cell migration. In the future, FOXA2 will be a novel biomarker for targeted therapy in esophageal cancer.

並列關鍵字

Esophageal cancer ； gene methylation ； cell migration

參考文獻

1. EA. M: World Cancer Report 2014: World Health Organization; 2014.

2. Siegel R, Ma J, Zou Z, Jemal A: Cancer statistics, 2014. CA: a cancer journal for clinicians 2014, 64(1):9-29.

3. 中華民國100年癌症登記報告. In.: Health promotion administration, Ministry of Health and Welfare; 2011.

4. Lee CH, Lee JM, Wu DC, Hsu HK, Kao EL, Huang HL, Wang TN, Huang MC, Wu MT: Independent and combined effects of alcohol intake, tobacco smoking and betel quid chewing on the risk of esophageal cancer in Taiwan. International journal of cancer Journal international du cancer 2005, 113(3):475-482.

5. Daly JM, Fry WA, Little AG, Winchester DP, McKee RF, Stewart AK, Fremgen AM: Esophageal cancer: results of an American College of Surgeons Patient Care Evaluation Study. Journal of the American College of Surgeons 2000, 190(5):562-572; discussion 572-563.