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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 賴亮全(Liang-Chuan Lai) | |
dc.contributor.author | Jun-Liang Luo | en |
dc.contributor.author | 羅俊良 | zh_TW |
dc.date.accessioned | 2021-06-17T06:10:54Z | - |
dc.date.available | 2019-03-05 | |
dc.date.copyright | 2019-03-05 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-11-09 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71818 | - |
dc.description.abstract | 缺氧是在一個在腫瘤微環境中典型的特徵,其對於癌症進程有深切的影響,而且與較差的預後密切相關。長片段非編碼核糖核酸(long non-coding RNA, lncRNA)是非編碼基因體的一員,由於其在腫瘤生成中扮演許多不同的角色而逐漸受到重視。先前我們實驗室使用次世代定序找到了在乳癌細胞株MCF-7中會被缺氧誘導表現的lncRNA,NDRG1-OT1,但是對於NDRG1-OT1的調控機制以及功能仍尚未釐清。因此本研究目的即為探討NDRG1-OT1在乳癌細胞中的轉錄機制以及可能扮演的功能角色。NDRG1-OT1的表現量分析顯示其在不同乳癌細胞株處於缺氧時皆會上升。而在過表現或者敲低缺氧誘導因子1α (HIF-1α)時分別會上調以及下調NDRG1-OT1的表現量。冷光酶報導基因分析法以及染色質免疫沉澱分析法證實了HIF-1α會結合到NDRG1-OT1的啟動子(-1,773到-1,769鹼基對及-647到-643鹼基對)上來活化其轉錄作用。接下來,為了探討NDRG1-OT1是否能作為吸附微小RNA (miRNA)的海綿,生物資訊工具首先被用來預測可能有交互作用的miRNA,而預測到的miRNA的表現量分析以及RNA免疫沉澱分析法的結果說明了NDRG1-OT1並不能作為miRNA海綿。最後關於NDRG1-OT1的功能角色方面,在常氧及擬缺氧狀況下過表現NDRG1-OT1並不會影響細胞增生、細胞遷移以及細胞週期的分布。總而言之,在這些結果中我們發現一個新的轉錄機制,即當乳癌細胞在缺氧刺激下,HIF-1α會透過轉錄活化而促進NDRG1-OT1的表現量上升。 | zh_TW |
dc.description.abstract | Hypoxia is a classic feature of tumor microenvironment, which has profound effects on cancer progression and is tightly associated with poor prognosis. Long non-coding RNAs (lncRNAs), a member of non-coding genome, have been increasingly investigated due to their diverse roles in tumorigenesis. Previously, our lab identified a hypoxia-induced lncRNA, NDRG1-OT1, in MCF-7 breast cancer cells using next-generation sequencing. However, the regulatory mechanism and functional roles of NDRG1-OT1 remain elusive. Therefore, the purpose of this study is to investigate the transcriptional mechanism and potential function roles of NDRG1-OT1 in breast cancer cells. Expression profiling of NDRG1-OT1 revealed that it was upregulated under hypoxia in different breast cancer cells. Over-expression and knockdown of HIF-1α up- and down-regulated NDRG1-OT1 respectively. Luciferase reporter assays and chromatin immunoprecipitation assays validated that HIF-1α transcriptionally activated NDRG1-OT1 by binding to its promoter (-1,773 to -1,769 and -647 to -643 bp). Next, to investigate whether NDRG1-OT1 could function as miRNA sponge, in silico analysis, expression profiling of predicted miRNAs, and RNA immunoprecipitation assays were performed, and the results implied that NDRG1-OT1 could not act as miRNA sponge. Lastly, regarding the function of NDRG1-OT1, ectopic expression of NDRG1-OT1 could not affect cell proliferation, migration, and cell cycle distribution under normoxia and hypoxia mimic conditions. In summary, our findings revealed a novel transcriptional mechanism of NDRG1-OT1 regulated by HIF-1α upon hypoxic condition in breast cancer cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T06:10:54Z (GMT). No. of bitstreams: 1 ntu-107-R05441017-1.pdf: 3142602 bytes, checksum: 54ef18c32fcec726857531f6d2bcc92e (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 致謝 I
摘要 II Abstract III Chapter 1 Introduction 1 1.1 Breast cancer and hypoxia 1 1.2 Emerging roles of long non-coding RNAs in cancer 2 1.3 Relationship between lncRNA NDRG1-OT1 and hypoxia 3 1.4 The aim of study 4 Chapter 2 Materials and Methods 6 2.1 Cell culture and treatments 6 2.2 Plasmid construction 7 2.3 Transfection and RNA interference 8 2.4 Site-directed mutagenesis 8 2.5 Luciferase reporter assay 9 2.6 RNA extraction, reverse transcription and quantitative RT-PCR 10 2.7 Nuclear-cytoplasmic RNA fractionation 11 2.8 Western blot 11 2.9 RNA immunoprecipitation (RIP) 12 2.10 Chromatin immunoprecipitation (ChIP) 13 2.11 Cell counting kit-8 (CCK-8) assay 14 2.12 Wound healing assay 14 2.13 Transwell migration assay 15 2.14 Colony formation assay 15 2.15 Cell cycle analysis 16 2.16 Statistical analysis 16 Chapter 3 Results 17 3.1 NDRG1-OT1 was upregulated under hypoxia in different breast cancer cell lines 17 3.2 Hypoxia inducible factors (HIFs) increased NDRG1-OT1 expression 17 3.3 Only HIF-1α regulated NDRG1-OT1 expression under hypoxia 19 3.4 HIF-1α transcriptionally activated NDRG1-OT1 under hypoxia by binding to its promoter 19 3.5 NDRG1-OT1 could not serve as miRNA sponge 21 3.6 NDRG1-OT1 could not affect cell functions of MCF-7 cells 22 Chapter 4 Discussion 24 4.1 The regulation of NDRG1-OT1 under hypoxia 24 4.2 Relationship between NDRG1-OT1 and miRNA sponge 26 4.3 Functional role of NDRG1-OT1 in breast cancer cells 28 4.4 Summary 29 Tables 30 Figures 36 References 55 Supplementary Data 64 | |
dc.language.iso | en | |
dc.title | 探討長片段非編碼核糖核酸NDRG1-OT1在乳癌細胞中的調控機制與扮演的功能角色 | zh_TW |
dc.title | Investigation of Regulatory Mechanisms and Functional Roles of Long Non-Coding RNA NDRG1-OT1 in Breast Cancer Cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 佘玉萍(Yuh-Pyng Sher),胡孟君(Meng-Chun Hu),蔡孟勳(Mon-Hsun Tsai) | |
dc.subject.keyword | 缺氧,長片段非編碼核糖核酸,NDRG1-OT1,乳癌,HIF-1a,微小RNA,常氧, | zh_TW |
dc.subject.keyword | Hypoxia,lncRNA,NDRG1-OT1,breast cancer,HIF-1a,miRNA,normoxia, | en |
dc.relation.page | 64 | |
dc.identifier.doi | 10.6342/NTU201804266 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-11-09 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生理學研究所 | zh_TW |
顯示於系所單位: | 生理學科所 |
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