雙酚A對於DNA甲基化之影響

Characteristics of bisphenol A affecting DNA methylation

黃瑋巧(Huang, Wei Chiao)

指導教授 ： 莊淳宇

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2011年)

https://doi.org/10.6843/NTHU.2011.00277

摘要

雙酚A (bisphenol A; BPA)是一種常見環境荷爾蒙，用做塑膠之塑化劑，具競爭雌激素受體(estrogen receptor; ER)之能力而影響人體內分泌，為內分泌干擾物質(endocrine disruptor)。BPA為脂溶性，有生物累積之特性，會囤積在人體脂肪細胞(adipose cells)中，可能影響脂肪激素(adipokines)分泌和能量代謝之恆定性而造成代謝性疾病。DNA甲基化(DNA methylatiom)為一種不改變基因序列而影響基因調控之方式。有文獻指出，BPA會造成異常基因甲基化，影響染色體的穩定性及基因表現，增加導致乳癌和子宮內膜癌的風險。
本論文研究目的有二，一、探討BPA對人類乳癌MCF-7細胞之存活率和脂肪激素(adipokines)－脂瘦素(leptin)和脂聯素(adiponectin)基因表現量之影響，及造成DNA序列甲基化之評估；二、探討懷孕婦女、子宮內膜增生婦女和不孕症婦女血液中BPA濃度和DNA甲基化之差異。實驗首先以不同濃度BPA (1, 10, 100, 103, 104, 105 nM)處理MCF-7細胞，以MTT assay (3-(4,5-dimethylthiazolyl -2)-2,5-diphenyltetrazolium bromide)檢測細胞存活率，以即時定量聚合連鎖反應(real-time quantitative polymerase reaction)檢測adiponectin及leptin之基因表現量。結果顯示1 nM之BPA會促進細胞之存活率， 104和105 nM之BPA會抑制細胞之存活率。此外，BPA會導致細胞週期S phase時期延長(10和105 nM)或縮短(103 nM)。 BPA (10、104和105 nM)會降低ER基因表現並誘導ER的表現。低劑量之BPA (10 nM)增加adiponectin基因表現、降低leptin基因表現；在103 nM BPA處理下，發現adiponectin基因表現上升但leptin之表現並無顯著降低；高劑量BPA (105 nM)增加adiponectin和leptin之基因表現。萃取全基因組DNA (genomic DNA)，利用sodium bisulfite處理後，以甲基化晶片(Illumina Infinium HumanMethylation27 BeadChip)偵測DNA甲基化位點，探究BPA暴露對於基因體甲基化之差異性及分布，以及影響基因之功能性分析，進而篩選受甲基化調控之基因。利用GO term分析甲基化晶片資料具有beta value及M value顯著差異的基因(n=22)，其與代謝、成長以及神經傳遞有關。其中，LIPE (hormone-sensitive lipase)是一參與代謝調控之基因，會受到adiponectin和leptin之影響，在Illumina甲基化測驗中發現BPA (10和105 nM)會導致LIPE去甲基化。基質金屬蛋白酶(matrix metalloproteinase; MMP)為鋅依賴之蛋白水解酵素，參與組織重塑，其啟動子活性受ER調控。本研究發現BPA會使MMP 13基因啟動子去甲基化，伴隨著mRNA表現減少。此外，在婦女樣本中也發現血液中BPA濃度和MMP13表現呈現負相關，可能藉由去甲基化所調控。
利用甲基化特異性PCR (methylation specific PCR; MSP)偵測婦女HOXA10 (homeobox A10)之甲基化程度和HOXA10 mRNA表現量之相關性。HOXA10基因為調控生長發育的重要因子，在子宮內扮演重要角色。懷孕婦女之HOXA10甲基化和mRNA表現呈負相關(r = -0.657)，而子宮內膜增生婦女之HOXA10甲基化和基因表現呈正相關(r = 0.8)。由本研究結果得知，BPA會導致異常之基因表現，其可能藉由甲基化及其他外基因體機制所調控。

關鍵字

雙酚A ； 雌激素受體 ； 脂瘦素 、脂聯素 ； DNA甲基化 ； 基質金屬蛋白酶13 ； HOXA10

並列摘要

Bisphenol A (BPA) is a common environmental estrogen as an endocrine disruptor to mimic endogenous estrogen 17-estradiol by binding to estrogen receptor. As lipophilicity and bioaccumulation, BPA has the potency to accumulate in adipose tissue and interfere with adipokin secretion and homeostasis of energy metabolism. BPA accumulates in adipose tissue might influence the gene expression of adiponectin and leptin leading to induce metabolic diseases. Due to common use for plastics products, human would expose low doses of BPA in daily life. Otherwise, BPA could cause abnormal DNA methylation affecting the stability of chromosome and gene expression to increase the risk of breast cancer and endometrial cancer. Thus, the purpose of this study was to investigate whether BPA caused cytotoxicity and gene expression in human breast cancer MCF-7 cells, and to explore the difference in DNA methylation and the BPA levels among pregnant women, women with endometrial hyperplasia (EH) and infertile women. The cell proliferation was increased in the 1 nM BPA treatment and attenuated at higher doses of BPA (104 and 105 nM). BPA changed the duration of S phase in cell cycle that prolonged at 10 and 105 nM BPA and shortened at 103 nM BPA. The results of real-time PCR showed that BPA treatment (10, 104 and 105 nM) attenuated the mRNA expression of ER and increased the expression of ER. With the treatment of 103 nM BPA, the gene expression of ER was increased as well as ER. Low-dose of BPA treatment (10 nM) increased the gene expression of adiponectin and decreased the expression of leptin. With the treatment of 105 nM BPA, MCF-7 cells had the increased expression of adiponectin and leptin. This study explored the distribution of DNA methylation and gene ontology to select candidate genes after BPA treatment using Illumina Infinium HumanMethylation27 BeadChip. Twenty-two genes selected from the difference of beta and M value were categorized into metabolic function, development and nervous process in analysis of GO term. LIPE (hormone-sensitive lipase) involved in the metabolic process was hypomethylated after BPA treatment (10 and 105 nM). MMP13 is a matrix metalloproteinase for tissue remodeling and its activity of promoter is regulated by ER. The results of this study showed that MMP13 had the hypomethylation level after BPA treatment in accompany with the low level of MMP13 mRNA expression.
Homeobox A10 (HOXA10) plays an important role in endometrium development. MSP (methyl specific PCR) was used in this study to determine the methylation level of HOXA10. This study found that a negative correlation between the methylation level of HOXA10 and its mRNA expression in pregnant women (r = -0.657), and a positive correlation in EH women (r = 0.8). In conclusion, these results indicated that BPA may influence the gene expression underlying DNA methylation and other epigenetic modulation.

並列關鍵字

BPA ； estrogen receptor ； leptin, adiponectin ； DNA methylation ； MM13 ； HOXA10

參考文獻

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