2015-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643946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，因腫瘤内的血管分布既沒效率且不穩定，所以 腫瘤内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，我們發現水敗67於覆氧後的表現量變 化最劇烈。過去文獻亦指出，NDRG1在許多環境壓力及生長受限的情況下、廣泛地表 現於許多組織中；因此我們假設光敗67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、淑况67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；在上次的計晝中，我們找 到芳香烴受體(aryl hydrocarbon receptor)為覆氧時調控NDRG1的轉錄因子。不過NDRG1 在覆氧時的生理功能仍不是很清楚。近年來因高解析度基因晶片及次世代定序技術的進展，許多無法被轉譯成蛋白質的 非編碼核糖核酸（noncoding RNA)相繼被發現，例如微型核糖核酸(microRNA)及長片 段非編碼核糖核酸（long non-coding RNA)。最近文獻亦指出這些非編碼核糖核酸參與 基因的調控。因此，我們假設NDRG1於覆氧時也受非編碼核糖核酸的調控，故提出此計 晝來詳細性地研究其作用機轉。我們提議進行下列實驗以達成的目標包括：第一、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；第二、研究氧濃度變化時，直接調控NDRG1的微型核糖核酸及其結合位置；第三、尋找氧濃度變化時，直接調控NDRG1的長片段非編碼核糖核酸。在我們初步的實驗中，我們證明覆氧時，NDRGJ能抑制乳癌細胞的生長及運動能 力；找到NDRG1可能是4個微型核糖核酸（miR-769-3p, miR-501-3p, miR-2276,及 miR-1282)的目標基因；運用生物資訊分析找到25個與氧變化相關的長片段非編碼核 糖核酸。總括而言，利用生物資訊分析、基因學技術、與功能測試，本計晝能讓我們更 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 is both inefficient and unstable. Although cellular adaptation to hypoxia is well documented, little is known about adaptive mechanisms to reoxygenation.In our previous genomic study of breast cancer cells upon reoxygenation, we identified that NDRG1 had the maximal response. NDRG1 is reported to be expressed ubiquitously in tissues stimulated under a wide variety of stress and cell growth-limited conditions. Therefore, we hypothesized that NDRG1 might 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 remained elusive. In our last proposal, we identified Aryl hydrocarbon receptor (AhR) as a novel transcription factor candidate regulating NDRG1. Yet, its physiological function under reoxygenation is still unclear.Recently, massive amount of novel non-coding transcripts were revealed, such as microRNA and long non-coding RNA, because of the advance of high-resolution microarray and next generation sequencing technology. Previous studies also reported that these non-coding RNAs participate in regulating gene regulation. Hence, we hypothesize that NDRG1 is regulated by these non-coding RNAs upon reoxygenation. Therefore, we propose to comprehensively investigate the regulatory mechanism of NDRG1 upon changes in oxygen concentrations by conducting experiments with the following specific aims:1)Investigation of functional roles of NDRG1 using in vitro and in assays;2)Investigation of microRNAs and their binding sites that directly regulate NDRG1 upon oxygen changes;3)Identification of long non-coding RNAs that directly regulate NDRG1 upon oxygen changes.In our preliminary results, we showed that NDRG1 can inhibit MCF-7 proliferation and migration under reoxygenation. Also, we have identified that NDRG1 may be the target gene of 4 miRNAs (miR-769-3p, miR-501-3p, miR-2276, and miR-1282). Using bioinformatics approach, we identified 25 oxygen responsive long non-coding RNAs. 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.覆氧NDRG1轉錄微型核糖核酸長片段非編碼核糖核酸功能性研究reoxygenationNDRG1transcriptionmicroRNAlncRNAlong non-coding RNAfunctional study