雙酚Ａ影響人類子宮內膜細胞microRNAs調控與癌化之關連性

Bisphenol A dysregulated microRNAs for carcinogenesis in human endometrial cells

李佩諠(Pei-Hsuan Lee)

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(2014年)

摘要

雙酚A (bisphenol A; BPA)是一種塑化劑，廣泛添加於聚碳酸酯(polycarbonate; PC)或環氧樹酯(epoxy)塑膠製品、牙科填充物，以及食品罐頭內層塗料。BPA為一種環境荷爾蒙，會與雌激素競爭雌激素受體，影響內分泌作用和細胞生長發育。子宮內膜增生為子宮內膜癌發生之危險因子之一。根據本研究室偵測結果，子宮內膜增生之婦女其血液中BPA含量較正常婦女為高；也發現BPA會誘發人類子宮內膜癌細胞(human endometrial cancer RL95-2 cells)發炎反應及促使細胞增生之基因表現。已有研究指出子宮內膜癌症患者有異常microRNAs (miRNAs)表現。因此，本研究欲探討BPA是否會影響miRNAs調控，改變基因表現，誘發人類子宮內膜細胞癌化反應。
本研究目的為：一、BPA暴露是否干擾miRNAs及其調控基因mRNAs之表現；二、評估BPA暴露影響之基因表現是否與子宮內膜癌患者中異常調節之基因功能有相關性。三、BPA暴露影響之miRNA調控基因路徑是否導致癌化。為找出BPA暴露可能造成子宮內膜癌之相關miRNAs及其目標基因，本研究分別以miRNA晶片(Phalanx Human miRNA OneArray® v4 chip)和mRNA晶片(Phalanx Human OneArray® v5 chip)，偵測人類子宮內膜癌細胞RL95-2 cells暴露BPA (10、103和105 nM)，導致具有表現差異之miRNA和mRNA (differentially expressed genes; DEG)。將BPA暴露之miRNA chip中有表現差異之miRNAs，對應以文獻回顧篩選人類子宮內膜癌癌化相關之miRNAs，並參照miRDB網站預測這些miRNA之目標基因，再對照mRNA chip找出受BPA暴露影響和人類子宮內膜癌癌化之目標DEG。以Kyoto Encyclopedia of Genes and Genomes database (KEGG)將DEG做功能分類，並以Cytoscape套裝軟體建構基因網絡分析。
從KEGG pathway和Cytoscape基因網絡分析顯示，BPA暴露會促使miR-149下調，影響DNA修復基因ARF6 (ADP-ribosylation factor 6)、TP53 (tumor protein p53)及CCNE2 (cyclin E2)表現，造成細胞週期改變。BPA暴露使miR-141和miR-205表現增加，下調腫瘤抑制基因，如PRC1 (polycomb repressor complex1)和SMAD2 (SMAD family member 2)。BPA暴露增加miR-107表現，會抑制hedgehog路徑中的負調節因子，如SUFU (suppressor of fused homolog)和GLI3 (GLI family zinc finger 3)，進而活化hedgehog路徑，促使癌化過程發生。進一步以miR-149 mimic轉染反證，上調miR-149時，BPA暴露後ARF6、TP53及CCNE2基因恢復正常表現，與對照組無顯著差異。轉染miR-107 inhibitor抑制miR-107時，BPA暴露對於SUFU和GLI3表現仍有下降趨勢，但下調程度減少。本研究發現BPA暴露影響miR-149、miR-141、miR-205及miR-107調控，造成細胞週期失調，影響DNA修復功能，造成Hedgehog訊息路徑傳導異常，可能誘發子宮內膜細胞之癌化發生。

關鍵字

雙酚A ； 子宮內膜癌 ； microRNA ； 基因網絡 ； DNA損傷 ； Hedgehog信號 ； 癌化

並列摘要

Bisphenol A (BPA) is a plasticizer wildly used in polycarbonate (PC) and epoxy plastic products, dental sealants and inner coating of cans. BPA is an endocrine disrupt compounds (EDCs) able to compete with estrogen for estrogen receptor (ER) binding to affect cell function. Endometrial hyperplasia is one of the risk factors to lead to endometrial cancer. Our previous study found endometrial hyperplasia patients have higher serum BPA levels than normal women, and BPA exposure induces inflammatory response and increases gene expression of cell proliferation in human endometrial cancer RL95-2 cells. Aberrant microRNAs (miRNAs) regulation has been identified in endometrial cancer (EC) patients. Therefore, this study assumed BPA exposure disrupted miRNA regulation and its gene expression in human endometrial cells corresponding to EC carcinogenesis.
The purposes of this study were (1) to investigate whether BPA exposure affected miRNAs and their target genes in human endometrial cells; (2) to assess whether BPA exposure affected gene functions relevant to EC; and (3) to figure out whether BPA exposure dysregulated miRNA-mediated pathways to lead to endometrial carcinogenesis. This study used Phalanx Human miRNA OneArray® v4 chip and Phalanx Human mRNA OneArray® v5 chip to respectively determine differentially expressed miRNAs and mRNAs (DEGs) in human endometrial cancer RL95-2 cells exposure to 10, 103 and 105 nM BPA. In addition, this study performed a literature survey to explore miRNAs relevant to human EC progression. The differentially expressed miRNAs both presented in the miRNA chip of BPA exposure and in the literature survey were selected to further predict the potential target genes using miRDB website. These potential target genes corresponded to DEGs in the mRNA chip of BPA exposure were the candidate DEGs regarding to BPA exposure and EC progression. The candidate DEGs were analyzed for ontology and gene network construction using KEGG (Kyoto Encyclopedia of Genes and Genomes database) and Cytoscape software.
According to the analysis of KEGG pathway and Cytoscape gene network, this study identified that BPA exposure reduced miR-149 expression to down-regulate miR-149-mediated DNA repair gene ARF6 (ADP-ribosylation factor 6), TP53 (tumor protein p53) and up-regulate CCNE2 (cyclin E2) to interrupt cell cycle. BPA induced miR-141 and miR-205 to down-regulate their target genes PRC1 (polycomb repressor complex1) and SMAD2 (SMAD family member 2) in tumor suppression. BPA also increased miR-107 and suppressed hedgehog signaling regulatory factors SUFU (suppressor of fused homolog) and GLI3 (GLI family zinc finger 3) to activate hedgehog signaling for the carcinogenesis. Furthermore, this study transfected miR-149 mimic and miR-107 inhibitor to confirm the effect of BPA exposure on miRNA regulation and gene expression. After miR-149 mimic transfection in RL95-2 cells, the gene expression of ARF6, TP53 and CCNE2 displayed no significant differences between BPA exposure and control. After miR-107 inhibitor transfection in RL95-2 cells, the gene expression of SUFU and GLI3 were mildly reduced in BPA exposure.
This study discovered that BPA exposure affected miR-141, -107, -205 and miR-149 to disrupt cell cycle arrest, suppress DNA repair system, and activate hedgehog signaling, which has been reported to participate in cancer development and metastasis. These findings provided an insight into the potential epigenetic mechanism of BPA exposure on the risk of endometrial carcinogenesis.

並列關鍵字

bisphenol A ； endometrial cancer ； microRNA ； gene network ； DNA damage ； hedgehog signaling ； carcinogenesis

參考文獻

21. Banno K, Kisu I, Yanokura M, Masuda K, Kobayashi Y, Ueki A, Tsuji K, Yamagami W, Nomura H, Susumu N et al: Endometrial Cancer and Hypermethylation: Regulation of DNA and MicroRNA by Epigenetics. Biochemistry research international 2012, 2012:738274.

1. Bergeron RM, Thompson TB, Leonard LS, Pluta L, Gaido KW: Estrogenicity of bisphenol A in a human endometrial carcinoma cell line. Mol Cell Endocrinol 1999, 150(1-2):179-187.

2. Camps M, Ruckle T, Ji H, Ardissone V, Rintelen F, Shaw J, Ferrandi C, Chabert C, Gillieron C, Francon B et al: Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis. Nat Med 2005, 11(9):936-943.

3. Dolinoy DC, Huang D, Jirtle RL: Maternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development. Proc Natl Acad Sci U S A 2007, 104(32):13056-13061.

4. Zhang Q, Xu X, Li T, Lu Y, Ruan Q, Lu Y, Wang Q, Dong F, Yang Y, Zhang G: Exposure to bisphenol-A affects fear memory and histone acetylation of the hippocampus in adult mice. Hormones and behavior 2014, 65(2):106-113.