雙酚A 透過microRNA 調控影響人類子宮內膜癌細胞之基因表現

雙酚A (bisphenol A; BPA)是一種常見的環境荷爾蒙，它會與雌激素競爭，干擾具有雌激素受體細胞的生長以及發育，擾亂人體內分泌系統。BPA 廣泛應用於塑膠類的生產，如嬰兒奶瓶，罐頭和牙科材料。在本研究室先前發現子宮內膜增生的婦女，其血液BPA 含量都較正常婦女高，並且子宮內膜增生婦女的HOXA10 (調控子宮內膜生長基因)表現量也較高。為瞭解BPA 暴露對於子宮內膜細胞生長之影響，本論文研究目的為：一、探討BPA 暴露對人類子宮內膜癌細胞之基因表現影響，建立相關路徑。二、探討BPA 暴露對miRNA 表現及其對調控基因之影響。本研究將人類子宮內膜癌細胞(human endometrial cancer RL95-2 cells)暴露在濃度10、103 和105 nM BPA，分別以基因晶片(Phalanx Human OneArray® v5 chip)和miRNA 晶片(Phalanx Human miRNA OneArray® v4 chip)偵測mRNA 和miRNA表現。基因晶片結果顯示，受到BPA 暴露影響之基因，功能分類包括細胞訊號傳導、傷口癒合，以及細胞增生和發育。其中，BPA 暴露會增加子宮內膜細胞發育及增生調控功能相關基因之表現，如PIK3CB (phosphoinositide-3-kinase)、IL-8 (interleukin 8)、CDKN1A (cyclin-dependent kinase inhibitor 1A)和Casp9 (caspase 9)。接著利用KEGG pathway 資料庫瞭解基因互相調控之上下游關係，發現這些改變表現之基因與調控細胞增生及發炎路徑有關。在致癌路徑中，上游基因PIK3CB 表現量上升會促使Casp9 和CDKN1A 蛋白質磷酸化，使子宮內膜細胞增生。本研究也發現BPA 暴露會影響RL95-2 細胞之miRNA 表現量。本研究挑出21個顯著受到BPA 暴露影響的miRNA，比對 miRDB 資料庫與基因晶片資料，顯示miRNA-1973 會與IL-8 基因序列互補結合。而在基因晶片和real-time PCR 分析結果均顯示BPA 暴露會增加IL-8 表現量、降低miRNA-1973 表現量。推論BPA 可能透過抑制miRNA-1973 表現而增加IL-8 基因表現。本研究探討BPA 暴露影響細胞的基因表現以及miRNA 的調控。BPA 會藉由調控hsa-miR-1973 的表現而引起IL-8 的表現，導致子宮內膜細胞RL95-2 發炎，且改變PIK3CB、CDKN1A 以及Casp9 基因表現而造成細胞癌化。

關鍵字

雙酚A ；雌激素受體；子宮內膜細胞； IL-8

並列摘要

Bisphenol A (BPA) is a common environmental hormone which competes with estrogen to disrupt human endocrine system. BPA is widely used to plastic production such as baby bottles, cans and dental composites. Human would expose low doses of BPA in daily life because common use for plastics products. Our laboratory previously found that the women with endometrial hyperplasia have the higher blood BPA levels than normal women as well as the HOXA10 gene expression (relevant to endometrial growth). In order to understand the impact of BPA for endometrial development, the purpose of this study was to investigate whether BPA caused abnormal gene expression in human endometrial cells and illustrate gene regulatory pathways, and to explore whether BPA would affect specific micro-RNA regulation corresponding to gene expression in endometrial cells. Human endometrial cancer (RL95-2) cells were treated with 10, 103 and 105 nM BPA, and determined the mRNA and miRNA expression respectively using Phalanx Human OneArray® v5 chip and Phalanx Human miRNA OneArray® v4 chip. The analysis of gene cluster and GO term described gene classification and gene function of chip data. The gene functions of changed expression were related to signal transduction, stress response, wound healing and proliferation. This study explored BPA induced the gene expression of PIK3CB (phosphoinositide-3-kinase), IL-8 (interleukin 8), CDKN1A (cyclin-dependent kinase inhibitor 1A) and Casp9 (caspase 9) in a dose-response manner. According to KEGG pathway, the changed gene expression in exposure BPA was illustrated to the regulation path of cell proliferation and inflammation. In cancer pathway, the elevated upstream PIK3CB increased phosphorylation of Casp9 and CDKN1A protein resulting in proliferation of endometrial cells underlying the occurrence of endometriosis. This study also found that BPA affected the expression of miRNA in RL95-2 cells. The study singled out 21 miRNAs statistical significantly affected by BPA exposure from III miRNA chip data, and used miRDB database for miRNA corresponding to the possible combination of mRNA. The sequence of miRNA-1973 can combine with IL-8 thereby inhibiting the expression of IL-8 gene. The results of gene chip and real-time PCR both showed BPA exposure increased IL-8 mRNA expression and reduced miRNA-1973 expression. It suggested that BPA exposure attenuated miRNA-1973 expression to increase the expression of IL-8. This study investigated BPA exposure influenced gene expression and miRNA regulation in human endometrial cancer cells. BPA would alter the expression of IL-8 by modulating hsa-miR-1973 expression for inflammation, and change the expression of PIK3CB, CDKN1A and Casp9 to process cells carcinogenesis.

並列關鍵字

bisphenol A ； estrogen receptor ； human endometrial cancer cells

參考文獻

Target in Endometrial Cancer. Clin Cancer Res.

differentially regulated by 17 beta-estradiol and diethylstilbestrol (DES). J Mol Biol 340,

U.K. (2012). Integration of MicroRNA databases to study MicroRNAs associated with

endometrioid cancers of the endometrium. Cancer Res 61, 4576-4582.

Marsit, C.J. (2010). Bisphenol A exposure leads to specific microRNA alterations in

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雙酚A 透過microRNA 調控影響人類子宮內膜癌細胞之基因表現

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