探討鄰苯二甲酸酯類影響大腸直腸癌腫瘤生成之相關基因網絡

Study on the potential gene network associated with phthalate exposure and tumorigenesis in colorectal cancer cells

蘇俊融(Jun-Rong Su)

指導教授 ： 廖憶純

國立臺灣大學/生命科學院/生化科技學系/碩士(2018年)

https://doi.org/10.6342/NTU201801933

摘要

鄰苯二甲酸酯類 (phthalates or phthalate esters) 為一種廣泛使用的塑化劑，容易被釋放至環境中，透過呼吸系統或飲食過程，直接被攝取而危害消化系統。Phthalate 亦為內分泌干擾物質，關於其暴露所造成之健康危害，大多著重在研究乳癌、前列腺癌、生殖毒性及兒童發育方面的影響，對於大腸直腸癌方面的研究較少，因此，闡明鄰苯二甲酸酯類對大腸直腸癌腫瘤生成的影響與機制為當務之急。本論文藉由巨量生物資料庫，同時分析與 phthalate 暴露及大腸直腸癌腫瘤生成之關鍵基因，並進一步使用大腸細胞株進行驗證，探討其可能之調控機制。本研究首先搜尋基因資料庫 ArrayExpress 與 Gene Expression Omnibus，分析臨床大腸癌原位、轉移及正常組織，和人類細胞暴露 phthalate 的基因表現數據，以線上平台 NetworkAnalyst 比對，找到差異表現基因 (differentially expressed genes; DEG)，接著，以分子網絡之整合性軟體 Cytoscape，進一步將 NetworkAnalyst 所找到之 DEG 重新進行關係串聯及視覺化，找到同時與 phthalate 暴露及大腸直腸癌發生與進程高度相關的關鍵基因與調控路徑。預測的基因網絡結果顯示，phthalate 暴露可能會影響以血管內皮生長因子 (vascular endothelial growth factor A; VEGFA) 為首之調控基因網絡，本研究進一步以人類正常大腸細胞株 CCD841 及大腸癌非轉移細胞株 SW480，驗證 diethylhexyl phthalate (DEHP) 對此預測訊息傳導路徑之影響，以即時定量 PCR 檢測基因表現的結果發現，上游目標基因 VEGFA、PDGFRB、SRC 及 AKT1等，在二種細胞株中皆如同預期在 DEHP 處理下有表現量上升的情況；而在 CCD841 中，下游目標基因 CDKN1A (p21) 及 CDKN1B (p27) 的表現，會隨著 DEHP 濃度的上升而逐漸提高，但在 SW480 中則沒顯著差異。另一方面，低濃度 DEHP (102 nM) 會使 SW480 細胞增生能力上升 1.2 倍，而高濃度 DEHP 則會抑制 CCD841 及 SW480 細胞增生的能力，高濃度 DEHP (106 nM) 還會使 CCD841 細胞移動能力增加 1.3 倍，但卻抑制 SW480 細胞移動能力。綜合本研究之結果顯示，DEHP 單獨暴露可能不會直接影響大腸細胞腫瘤發生以及加劇大腸直腸癌的進程。

關鍵字

大腸直腸癌 ； 鄰苯二甲酸酯類 ； 巨量生物資料庫 ； ArrayExpress ； NetworkAnalyst ； 差異表現基因 ； Cytoscape ； 調控路徑 ； 血管內皮生長因子

並列摘要

Phthalate or phthalate esters (PAEs) are widely used as plasticizers, which can be easily released into the environment. Phthalate can be directly absorbed through respiratory or digestive system. Phthalates are also endocrine-disrupting component and several studies regarding the effects of phthalate exposure are mainly focused on breast cancer, prostate cancer, reproductive toxicity and child development. However, the health effects of phthalate exposure on colorectal cancer are rarely discussed. Therefore, it is urgently needed to clarify the effect and mechanism of phthalates on colorectal tumorigenesis. In this study, emerging high-throughput omics technology is used to analyze the critical hub genes associated with phthalate exposure and colorectal tumorigenesis. The role of the critical hub genes in the predicted regulatory pathway are further validated using CRC cell lines exposed to phthalate. First, we analyze the gene expression database and compare the gene expressions of clinical primary CRC (pCRC), metastatic CRC (mCRC), normal colon tissues and human cells with phthalate exposure from ArrayExpress and Gene Expression Omnibus. Then NetworkAnalyst is used to determine the differentially expressed genes (DEG) in phthalate exposure (phthalate exposure vs. non-exposure), pCRC (primary CRC vs. normal colon) and mCRC (metastatic CRC vs. primary CRC). Furthermore, we establish and visualize the potential network to identify the potential hub genes and regulatory pathways associated with phthalate exposure in CRC by Cytoscape. The gene network shows that phthalate exposure may affect the vascular endothelial growth factor A (VEGFA)- mediated regulatory pathways. We next verify the predicted potential regulatory pathways using normal colon cell line CCD841 and non-metastasis colon cancer cell line SW480 treated with diethylhexyl phthalate (DEHP). Real-time PCR reveals that upstream regulatory genes, VEGFA, PDGFRB, SRC and AKT1, are up-regulated in the both cell lines. DEHP induces the expression of downstream regulatory genes, CDKN1A (p21) and CDKN1B (p27) in CCD841 cells whereas there is no difference in SW480 cells. On the other hand, the low dose (102 nM) of DEHP enhances 1.2-fold proliferative ability in SW480 cells and high dose (106 nM) of DEHP inhibits the proliferation in both cell lines. Treatment of 106 nM DEHP results in a 1.3-fold increase but decreases in cell migration in CCD841 and SW480 cells, respectively. To sum up, this study suggests that DEHP alone may not directly affect colorectal tumorigenesis and CRC progression.

並列關鍵字

phthalates ； colorectal cancer ； meta-biological database ； ArrayExpress ； NetworkAnalyst ； differentially expressed gene ； Cytoscape ； regulatory pathway ； vascular endothelial growth factor

參考文獻

李家偉 (2008)，室內鄰苯二甲酸酯類暴露與兒童健康之相關性--子計畫 四：室內含鄰苯二甲酸酯類之民生消費品逸散特性及影響因子之研究，行政院國家科學委員會專題研究計畫。

劉梁明琦 (2016)，以巨量基因資料分析雙酚A對於大腸癌癌化之潛在路徑並探究大腸癌轉移之目標基因，碩士論文，國立清華大學，生醫工程與環境科學系。

衛生福利部國民健康署 (2015) 癌症登記年報。

Adir M, Combelles CMH, Mansur A, Ophir L, Hourvitz A, Orvieto R, Dor J, Machtinger R. Dibutyl phthalate impairs steroidogenesis and a subset of LH-dependent genes in cultured human mural granulosa cell in vitro. Reprod Toxicol. (2017) 69:13-18.

Albro PW, Corbett JT, Schroeder JL, Jordan S, Matthews HB. Pharmacokinetics, interactions with macromolecules and species differences in metabolism of DEHP. Environ Health Perspect. (1982) 45:19-25.