2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643948摘要：在過去數十年中，缺氧一直是受矚目的研究課題。因為研究中發現，缺氧腫瘤較不 易被治療且病人的預後較差。但實際上，缺氧腫瘤細胞內、氧濃度是一直變異的；因腫 瘤內的血管分布既沒效率且不穩定，使腫瘤內缺氧的區域常會因血液再灌流而重新覆 氧。過去數篇研究也曾報導癌細胞經缺氧並覆氧的刺激後，會增加其抗藥性及轉移的能 力。因此，因缺氧/覆氧常伴隨出現，故我們必須將缺氧與覆氧視為一體兩面的細胞刺 激。但過去僅專注於細胞對缺氧的研究，細胞對覆氧的適應機轉仍知道有限。先前利用乳癌細胞於覆氧的基因體研究中，我們發現 127 個基因參與此反應。傳導 路 徑 分 析 中 亦 發 現 這 些 對 氧 濃 度 變 化 有 反 應 的 基 因 中 ， 主 要 參 與 的 傳 導 路 徑 為 HIF-1-alpha 轉錄因子調控路徑與 C-MYC 轉錄因子啟動的下游基因。而其中，NDRG1 基 因於覆氧後的變化最劇烈，並且此基因又受 MYC 訊息傳導路徑所調控，故我們假設 NDRG1 基因於癌細胞適應氧濃度變異中、扮演一重要角色。雖然過去研究曾指出、NDRG1 基因 可於缺氧的環境下表現，且與癌細胞轉移的能力相關。但調控 NDRG1 基因的詳細機轉與 其在覆氧時所扮演的角色仍不明瞭。因此，我們計畫全面性地研究 NDRG1 基因於氧濃度 變化時的詳細調控機轉。為證明所提的假設，我們提議進行下列實驗以達成的目標包括：第一、尋找直接調控 NDRG1 基因的轉錄因子及其結合位置； 第二、尋找直接調控 NDRG1 基因的微型核糖核酸及其結合位置； 第三、研究 NDRG1 基因的調控是否受其啓動子中甲基化程度影響；第四、利用癌細胞的體外實驗及裸鼠實驗模型來探討 NDRG1 基因於生理功能上所扮 演的角色。利用基因學技術、功能測試與生物資訊分析，本計劃能讓我們更深了解 NDRG1 基因， 在癌細胞面對不同氧濃度變化環境中、在基因轉錄及生理功能上所扮演的角色。藉由更 清楚地了解癌細胞如何適應其周圍環境的分子機轉，有助於我們發展針對腫瘤惡化的治 療方式。<br> Abstract: Hypoxia has been intensively investigated over the past decades based on theobservations that hypoxic tumors were more resistant to therapy and had a worse prognosis. However, the oxygen concentration within hypoxic tumors was highly variable, because tumor vasculature was both inefficient and unstable. The hypoxic regions could become rapidly re-perfused or re-oxygenated. Several studies have been reported that tumor cells displayed increases in drug resistance and metastatic potential after exposed to hypoxia/reoxygenation insults. Therefore, it is necessary to consider hypoxia and reoxygenation as two parts of the same stress response as they were inevitably associated with each other. Although cellular adaptation to hypoxia was well documented, little was known about adaptive mechanisms to reoxygenation.In our previous genomic study on breast cancer cells upon reoxygenation, we identified127 genes involved in this response. Pathway analysis revealed that these oxygen-responsive genes were enriched in HIF-1-alpha transcription factor network, and validated targets of C-MYC transcriptional activation. Among these differentially expressed genes upon reoxygenation, NDRG1 had the maximal response and was regulated by MYC signalling pathway. Therefore, we hypothesize that NDRG1 may play an important role in tumor adaptation to fluctuation of oxygen concentrations. Although several studies suggested that NDRG1 is induced by hypoxia and associated with metastasis, the regulatory mechanism of NDRG1 remains elusive and its function under reoxygenation is still unclear. Hence, we propose to comprehensively investigate the regulatory mechanism of NDRG1 upon changes in oxygen concentrations. In order to prove our hypothesis, we propose to conduct experiments with the following specific aims:1) Identification of transcription factors that directly regulate NDRG1 and their binding sites;2) Identification of microRNAs that directly regulate NDRG1 and their binding sites;3) Investigation of methylation status in the promoter of NDRG1 regulating its expression;4) Investigation of functional roles of NDRG1 using in vitro and in vivo assays.Using genetic approaches, functional assays, and in silico analysis, this study will allow us to get a deep insight of the genetic mechanisms and functional roles of NDRG1 upon oxygen variation in transformed cells. By a better understanding of the molecular mechanism that cancer cells adapt to the tumor microenvironment, we hope to contribute in developing a more specific therapeutic regime to treat cancer.氧NDRG1轉錄轉錄微型核糖酸甲基化功能性研究oxygenNDRG1transcriptionmicroRNAmethylationfunctional study