在肺癌細胞株H1299探討EMT指標vimentin之調控機制：多環芳香烴受體之角色探討

Studies on the Mechanism of Regulation in EMT marker Vimentin in H1299：The Role of Aryl Hydrocarbon receptor

楊于萱(Yu-Hsuan Yang)

指導教授 ： 康照洲

國立臺灣大學/醫學院/毒理學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.01328

摘要

多環芳香烴受體 (Aryl hydrocarbon receptor, AhR) 是一配體活化之轉錄因子，當接觸到環境汙染物多環芳香烴 (polycyclic aromatic hydrocarbon, PAH) 時多環芳香烴受體 (Aryl hydrocarbon receptor, AhR) 會被活化並啟動下游基因表現。細胞上皮-間質轉換 (epithelial - mesenchymal transition, EMT) 是指在生理上胚胎發育及癌症發展過程中，細胞失去上皮細胞緊密結合型態的過程。EMT不僅促進胚胎發育過程中型態改變，在癌症中更促使癌細胞侵襲及轉移。過去文獻報導，多環芳香烴苯芘 (benzo[a]pyrene, B[a]P) 會藉由活化AhR，造成細胞中許多EMT指標在mRNA的表現呈現促進EMT現象。此外，亦有文獻指出，AhR在未與任何PAH結合活化的情形下，與調節細胞骨架重組、細胞型態及爬行能力有關。然而，AhR在未活化的狀態下與EMT之間的關係目前還是未知。因此，本研究目的在探討AhR於EMT機轉當中所扮演的角色。首先，從A549、H1299及CL1-5、CL1-0兩組肺癌細胞株發現，當細胞AhR表現量多時細胞侵襲能力小，而AhR表現量少則細胞侵襲能力大，後續我們選取AhR含量較少之H1299細胞做AhR大量表現載體 (AhR overexpressing vector) 的暫時性轉染，發現AhR大量表現之H1299其細胞型態呈現較為狹長且侵襲能力下降。利用核質分離及免疫螢光染色發現大量表現之AhR多分布於細胞質中，而處理AhR配體苯芘 (Benzo[a]pyrene) 後AhR進核並可誘導下游基因CYP1B1的產生。進一步探討作用機轉其對EMT指標之影響，發現間質指標 (mesenchtmal marker)─vimentin在AhR大量表現之劑量及時間效應實驗上蛋白質表現量有顯著下降，但mRNA表現量不受影響，此外，vimentin上游GTPases家族中之Rac1亦只在蛋白質表現量有下降現象，顯示AhR可能是透過轉譯後修飾對蛋白質進行調節作用。然而，Rac1抑制劑NSC23766處理無法回復由AhR大量表現所造成之vimentin抑制，但仍可看到磷酸化型式之vimentin有上升現象，可知AhR大量表現所引發的vimentin抑制並非經由Rac1所調控。另一方面，處理MG132後可回復AhR大量表現造成之Rac1及vimentin抑制，進而利用免疫沉澱法發現當AhR大量表現時AhR與Rac1、Ubiquitin、vimentin及phospho-vimentin之間交互作用皆有增之加現象。

關鍵字

多環芳香烴受體 ； 上皮間質轉移 ； vimentin ； Rac1

並列摘要

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. It will translocate into nucleus and may directly linked to transcriptional activation of target genes in response to ligands, polycyclic aromatic hydrocarbon (PAH). Epithelial - mesenchymal transition (EMT) is a process in which epithelial cells lose its intercellular adhesion during critical phases such as embryonic development and tumor progression. Previous studies have shown that EMT will be induced by ligand-activated AhR through activating a transcription factor of EMT. However, in our study, the non-activated AhR could decrease EMT in H1299 cell, and the detailed mechanism is still unknown. In this study, we found that the level of AhR expression in different lung cancer cells was correlated with their invasive potential. H1299 cell, which expressed AhR in low basal level, showed a high expression level in vimentin, an EMT marker, and showed a higher invasive ability (compared with A549, lung epithelial cell with high-level AhR expression and lower invasive ability). When AhR overexpression in H1299, not only the expression of vimentin was reduced, but the invasive cell was suppressed. Although the vimentin protein level was changed in AhR overexpressed cell, whereas the mRNA level was remained unaffected. We also found the overexpressed AhR protein was stayed in cytosol, and without transcriptional activity until the stimulation of B[a]P, suggested the anti-EMT function of AhR was independent of their transactivational activity. Moreover, non-activated AhR reduced the protein expression level of GTPase exchange factor (GEF), Rac1, and without change in their mRNA level. However, treatment of Rac1 inhibitor NSC23766 did not reverse the decrease in vimentin, and the phospho- vimentin still increased, suggested that overexpressed-AhR regulate vimentin through Rac1-independent pathway. Moreover, we found that the protein-protein interaction between AhR and Rac1, Ubiquitin, vimentin, phospho-vimentin was increased in AhR-overexpressed H1299 cell.

並列關鍵字

Aryl hydrocarbon receptor (AhR) ； Epithelial mesenchymal transition (EMT) ； vimentin ； Rac1

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