以 RNA 干擾技術探討人類肺腺癌細胞中戴奧辛受器對癌症相關之基因表現與細胞生長的影響

Utilizing RNA interference technique to investigate effect of aryl hydrocarbon receptor on expression of cancer-related genes and cell growth in human lung adenocarcinoma

陳柏宏(Po-Hung Chen)

指導教授 ： 蔡菁華 ； 林嬪嬪

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2006年)

https://doi.org/10.6834/CSMU.2006.00001

摘要

台灣地區從民國 71 年起，癌症就是民眾死因的首位。由衛生署民國 93 年的統計資料中可知，肺癌已成為男性癌症死亡原因的第二位，而在女性已是超越子宮頸癌成為第一位。由許多報告與流行病學的資料指出暴露環境化學毒物 (如戴奧辛) 是肺癌的重要致病因子之一，也證實長期暴露於這些物質的環境中會增加民眾罹患肺癌的危險性。TCDD 是戴奧辛受器 (Aryl hydrocarbon receptor ，AhR) 的配基，標的細胞暴露於這些配基後能使細胞質中的 AhR 活化，進而促進細胞色素 P450 1 (CYP1) 基因表現，已知這些酵素能代謝活化產生致癌性的中間產物，使細胞進行癌化。本實驗室先前研究發現，肺癌患者肺組織中的腫瘤細胞的 AhR 蛋白量比正常細胞多，且肺腺癌細胞株也比正常肺細胞株表現較高量的 AhR，故推測 AhR 可能與肺腺癌的發生有關。因此本論文的目的為：(1) 以 RNA 干擾技術，抑制肺腺癌細胞株 H1355 中 AhR 的表現，探討 AhR 在肺腺癌細胞中扮演的生理角色，(2) 探討 AhR 對其所調控 CYP1 酵素的基本表現 (constitutive expression) 的影響。我們首先建構一個 AhR RNAi 的 DNA 載體，轉染至 H1355 細胞株並經 G418 篩選後，挑選能持續穩定地表現 AhR RNAi 的 stable clones，以定量即時同步聚合鏈鎖反應 (RT real-time PCR) 及西方墨點法確定 stable clones 中的 AhR RNA 和 protein 表現受到抑制的程度分別達 80﹪ 及 70％。並且發現 AhR 下游調控基因 CYP1B1 在基因及蛋白層次表現會受 AhR 表現影響，但 CYP1A1 則否。而藉由 MTT assay、Anchorage independent colony formation assay 及流式細胞儀測定 ROS，發現 AhR 表現多的細胞其colony formation 的能力較強、ROS 產生也較多，由以上結果推論，AhR 可能與增加癌細胞的生長有關。另一方面，我們利用 Human cancer pathway microarray 發現 TSP1 及 MTS1 (S100A4) 表現量與細胞 AhR 表現量呈現正相關；而在 Transcription Factor Protein / DNA Array 中亦發現許多轉錄因子與 DNA binding 活性會隨 AhR 表現改變而變動，如 ER（estrogen receptor）、NF-1（nuclear factor 1）、Oct-1（POu domain, class 2, transcription factor 1）等與細胞 AhR 表現量呈現正相關，而 PR（progesterone receptor）則與細胞 AhR 表現量呈現負相關。再者我們也在蛋白層次上看到 AhR 會影響某些細胞蛋白表現，14-3-3 gamma、Galectin-1 二個與抑制癌細胞生長相關的蛋白及 HSP27，在 AhR 表現較低的細胞中表現較高；而 GRB10 則在 AhR 表現較低的細胞中表現較低。從 Human cancer pathway microarray 分析的結果與細胞蛋白測定的結果來看，我們確信 AhR 在 tumorigenesis 過程中著實佔有重要的角色。

關鍵字

戴奧辛受器 ； 人類肺腺癌細胞

並列摘要

Malignant neoplasms have been the leading cause of deaths in Taiwan since 1982. In 2004, lung cancer is the first and second leading cause of cancer death among females and males in Taiwan, respectively. Epidemiological studies indicated that exposure to environmental carcinogens (such as dioxins ) might be one of etiologies for human lung cancer, and increase the risk of lung cancer. 2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is ligand of aryl hydrocarbon receptor (AhR). Liganded AhR up-regulates expression of cytochrome P450 1 (CYP1) enzymes in target cells. Our previous studies indicated that AhR expression was increased in human lung adenocarcinomas (AD) tissues and cell lines. It suggested that AhR might play an important role in the development of lung adenocarcinomas. There are two objectives in the present study: 1) to evaluate the physiologic role of AhR in lung AD cells with the RNAi technique, 2) to study effect of AhR expression on constitutive expression of CYP1A1 and CYP1B1 in lung AD cells. We created a DNA vector containing human HU6 promoter followed by the AhR siRNA sequence. This construct was transfected into lung AD cell lines H1355 and G418 resistant stable clones were selected. The quantitative real-time PCR and Western immuno-blotting analysis showed that AhR mRNA and protein levels in these stable clones were respectively inhibited up to 80% and 70%. We found that AhR expression affected CYP1B1 mRNA and protein expressions, but not CYP1A1. Utilizing MTT assay, anchorage independent colony formation assay and flow cytometry for measuring intracellular ROS, we found that cells with AhR high expression formed more colonies in soft agar and generated more intracellular ROS than those with low AhR expression. Therefore, AhR might promote cancer cell growth. On the other hand, we found that TSP1 and MTS1 (S100A4) expressions were correlated with AhR by Human cancer pathway microarray assay. Utilizing Transcription Factor Protein / DNA array, we found that activity of many transcriptional factors for DNA binding was modified by AhR. For example, estrogen receptor (ER), nuclear factor 1 (NF-1), POU domain, class 2, transcription factor 1 (Oct-1) et al. were correlated with AhR, and progesterone receptor (PR) was inversely correlated with AhR expression. Furthermore, we also found that AhR expression affected some proteins expression. 14-3-3 gamma, Galectin-1 and Hsp27 expressions were inversely correlated with AhR expression, and GRB10 expression was correlated with AhR expression. Taken together, our results suggested that AhR might play an important role in the tumorigenesis.

並列關鍵字

Aryl hydrocarbon receptor ； human lung adenocarcinoma

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