三甲基膽蒽對缺氧反應元件之影響： 多環芳香烴受體之角色探討

Abstract

多環芳香烴（PAHs）為廣泛存在的環境污染物，3-甲基膽蒽（3-methylcholanthrene,3-MC）屬於多環芳香烴中的一員，當3-甲基膽蒽活化多環芳香烴受體（aryl hydrocarbon receptor, AhR）後會與芳香烴受體核轉位蛋白（AhR nuclear translocator, ARNT, HIF-1β）形成複合體然後啟動下游代謝解毒酵素CYP1A1 等基因的轉錄活化。缺氧反應元件（hypoxia-responsive element, HRE）主要是藉由缺氧誘導因子-1α（hypoxia-inducible factor-1 alpha, HIF-1α）所調控，然而過去研究發現利用小鼠Cl41 細胞處理多環芳香烴或其衍生物會使HRE下游基因血管內皮成長因子（vascular endothelial growth factor, VEGF）的mRNA 表現上升，此外文獻也觀察到3-MC 處理小鼠primary hepatocytes 後並不會對缺氧誘導的基因造成改變。因此本篇目的欲探討多環芳香烴影響細胞HRE 活性的相關機制，本研究採用三種不同的細胞株作為實驗材料293T、GBM 及HepG2，進行暫時性轉染HRE reporter gene 後，分別處理不同濃度的3-甲基膽蒽後藉由HRE promoter reporter assay 觀察其活性變化，結果顯示293T 和GBM 細胞的HRE 具有活化的現象而在HepG2細胞中則否。而實驗進一步發現HepG2 細胞的AhR：ARNT 比值遠大於293T 和GBM細胞，然而此差異為AhR 蛋白的含量不同而造成的，因此本研究假設3-MC 造成此三株細胞的HRE 活性變化的現象可能與AhR 相關。此外利用RT-PCR 觀察293T 與HepG2細胞處理3-MC後其HRE下游基因VEGF 的mRNA表現量，在293T 細胞中VEGF mRNA表現量會上升而HepG2 細胞中則否；並且在西方墨點法分析中，發現293T 及GBM 細胞處理3-MC後並不會造成其HIF-1α 蛋白產生穩定累積的現象。本研究進一步利用AhRshRNA 使HepG2 細胞的AhR 靜默（silence）探討其扮演的相關角色，實驗發現AhR 靜默的HepG2 細胞處理三甲基膽蒽後，能誘導其HRE 活性上升，並且在與缺氧狀態下處理三甲基膽蒽後其HRE活性反應更強烈；而利用免疫沉澱法觀察發現AhR靜默的HepG2 細胞其HIF-1α與ARNT之間的鍵結力，而造成細胞中HRE活性改變。

Polycyclic aromatic hydrocarbons (PAHs) are common environmental contaminants. 3-Methylcholanthrene (3-MC) belongs to a group of PAHs which is an aryl hydrocarbon receptor (AhR) agonist. 3-MC binds to AhR and subsequent translocates to the nucleus to activate the downstream gene expression, such as CYP1A1. Hypoxia-responsive element (HRE) are activated mainly by hypoxia-inducible factor-1alpha (HIF-1α) under hypoxia condition, but the present study found that exposure of cells to PAHs and its derivatives led to marked induction of VEGF in Cl41 cells, and other study in mice primary hepatocytes have shown that exposure to PAHs, 3-MC, did not significantly alter HIF target gene expfected hypoxia reporter) and increased HRE downstream gene VEGF mRNA level in 293Tression. In this study, we found that 3-MC can induce HRE activity (a transiently trans and GBM cells, but 3-MC had no effect on a transiently transfected hypoxia reporter in HepG2 cells. We also found that 3-MC-induced HRE activity in GBM and 293T cells is independent of HIF-1α. We observe HepG2 cells has a high level of AhR protein, so we thought that the differences may be due to the differences of AhR : ARNT ratios in the cells. In addition, we used AhR shRNA to silence the AhR of HepG2 cells, and we subsequent found that 3-MC can induced HRE activity in HepG2 AhR knockdown cells, and 3-MC can enhance HRE activity in HepG2 AhR knockdown cells under hypoxia. Besides, we also used immunoprecipitation assay to observe AhR, HIF-1α and ARNT interaction. In this experiment we observed that the AhR can inhibited HIF-1α-ARNT interactions. Therefore, the results suggest that AhR may affect HRE activity due to the decreased availability of ARNT for HIF-1α dimerization.