2013-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/660050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，因腫瘤內的血管分布既沒效率且不穩定，所以腫瘤內缺氧的區域常會因血液再灌流而重新覆氧。但過去研究大多專注於細胞對缺氧的研究，細胞對覆氧的適應機轉所知道仍非常有限。 先前利用乳癌細胞於覆氧時的基因體研究中，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 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 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 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 的詳細機轉與其在覆氧時所扮演的角色仍不明瞭。因此，我們計畫詳細性地研究 NDRG1 於氧濃度變化時的調控機轉。 根據審查者對先前計畫的意見及建議，在本次修改的計畫中，我們已加入許多初步的實驗結果並將目標濃縮來證明我們所提的假設。我們提議進行下列實驗以達成的目標包括： 第一、鑑定氧濃度變化時，芳香烴受體（aryl hydrocarbon receptor）是直接調控 NDRG1的轉錄因子； 第二、尋找氧濃度變化時，直接調控 NDRG1 的微型核糖核酸及其結合位置； 第三、利用癌細胞的體外實驗及裸鼠實驗模型來探討 NDRG1 於生理功能上所扮演的角色。 在我們初步的實驗中，我們找到芳香烴受體(aryl hydrocarbon receptor)可能為調控NDRG1 的轉錄因子。再者，我們也找到 NDRG1 可能是覆氧時、4 個微型核糖核酸（miR-769-3p, miR-1282, miR-501-3p, and miR-2276）的目標基因。先前我們亦證明降低NDRG1 能促進乳癌細胞的轉移能力。總括而言，利用生物資訊分析、基因學技術、與功能測試，本計畫能讓我們更深了解 NDRG1，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 the observations that hypoxic tumors were more resistant to therapy and had a worse prognosis. However, the hypoxic regions could become rapidly re-perfused or re-oxygenated, because tumor vasculature was both inefficient and unstable. Although cellular adaptation to hypoxia was well documented, little was known about adaptive mechanisms to reoxygenation. In our previous genomic study of breast cancer cells upon reoxygenation, we identified 127 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. Based on reviewers’ comments and suggestion to previous proposal, in this extended study, we have included more preliminary data and condense our specific aims to prove our hypothesis. We propose to conduct experiments with the following specific aims: 1) Identification of aryl hydrocarbon receptor as the transcription factor that directly regulates NDRG1 upon oxygen changes; 2) Identification of microRNAs and their binding sites that directly regulate NDRG1 upon oxygen changes; 3) Investigation of functional roles of NDRG1 using in vitro and in vivo assays. In our preliminary results, we have identified AhR (Aryl hydrocarbon receptor) as a novel transcription factor candidate regulating NDRG1. Also, NDRG1 may be the down-stream gene of 4 miRNAs (miR-769-3p, miR-1282, miR-501-3p, and miR-2276). Previously, we also showed that down-regulation of NDRG1 correlates with increase of MCF-7 migration under reoxygenation. In summary, using in silico analysis, genetic approaches, and functional assays, this study will allow us to get a deeper 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.