臺灣博碩士論文加值系統

氧分壓的不穩定是腫瘤微環境的特色之一，復氧引起的氧化壓力(oxidative stress)導致細胞內遺傳物質的損壞(DNA damage)和不穩定(genomic instability)。在缺氧、復氧的環境中篩選存活下來的癌細胞，其惡性(malignancy) ， 藥物抗性(drug resistance) ， 以及轉移能力(metastatic potential)都被證實有明顯的增強。我們認為癌細胞在缺氧後復氧會引發數種細胞反應(cellular response) 讓癌細胞得以因應這樣的環境並且存活。將較不具侵略性的乳癌細胞株MCF-7 在0.5%的低氧環境下培養24 小時後復氧0、1、4、8、12 和24 小時，進行基因體的基因表現量微陣列(gene expression microarray)分析。篩選出有顯著變化的274 個基因，其中47.7%的基因表現有顯著的增加；53.2%表現量減少。進行Ingenuity Pathway Analysis 找出顯著的canonical pathways 並建立基因網絡(genenetwork)。篩選分析後發現CCND3和NDRG1 在復氧後找到的十個後選基因中表現量差異最大，實驗顯示復氧會重新啟動缺氧以及抑制CCND3基因表現導致的細胞週期停滯，而在復氧下大量表現NDRG1基因則會抑制細胞的行為能力主要是透過過氧化分子的調控。研究除了提供MCF-7 在缺氧後復氧的基因表現和分子途徑(molecular pathways)的動態變化，同時增進了腫瘤細胞對復氧反應的了解。

Oxygen fluctuation resulting from hyper-proliferation of tumor cells, abnormal metabolism and disorganized tumor neovasculature characterizes the microenvironment of many cancers, which influences tumor development and angiogenesis. Hypoxia/ reoxygenation induce oxidative stress, which leads to DNA damage and genomic instability. Multiple cellular responses were activated in order to survive under this microenvironment, but little is known about the dynamic response upon reoxygenation. To investigate the dynamic response of signalling pathways in tumor adaptation, a breast cancer cell line MCF-7 was cultured under 0.5 % oxygen condition 24 hours and was harvested at various time points during reoxygenation. Genome-wide microarray results revealed that 274 genes were differentially expressed during reoxygenation; 47.4% of them were up-regulated and 52.3% down-regulated. Furthermore, pathway analysis, including canonical pathway and gene networks, were performed using Ingenuity Pathway Analysis. Selected genes of interest were validated by quantitative real-time PCR, such as N-myc downstream regulated 1 (NDRG1) and cyclin D3 (CCND3). Reoxygenation restored G1 arrest induced by CCND3 knockdown compared to hypoxia. Overexpression NDRG1 suppressed cell migration specifically under reoxygenation through ROS regulating NDRG1 expression in MCF-7. Our results showed dynamic changes of MCF-7 gene expression and molecular pathways upon reoxygenation, which shed some light on understanding cellular response to reoxygenation.

Table of Contents
Acknowledgements iii
Chinese Abstract v
English Abstract vi
Table of Contents vii
List of Figures viii
List of Tables viii
Chapter 1 Introduction 1
1.1 Tumor microenvironment and carcinogenesis 1
1.2 Hypoxia 3
1.3 Reoxygenation 6
1.4 Preview of this study 7
Chapter 2 Materials and Methods 9
2.1 Cell lines and cell culture 9
2.2 Sample preparation 9
2.3 Illumina human whole-genome expression analysis 10
2.4 Data mining and statistical analysis 10
2.5 Expression construct and generation of NDRG1 overexpressed cells of MCF-7 11
2.6 siRNA-mediated silencing of CCND3 12
2.7 Protein extraction and immunoblotting 12
2.8 Reverse transcription and quantitative Real-Time PCR 13
2.9 Cell migration assay 14
2.10 Cell cycle analysis 15
2.11 ROS scavenger treatment 15
Chapter 3 Result 16
3.1 Dynamic Response of MCF-7 in Responsive to Reoxygenation 16
3.2 Temporal Analysis of Gene Expression During Reoxygenation After 24h of Hypoxia 17
3.3 Canonical Pathways Responsive to Reoxygenation. 18
3.4 Representative Pathways of Each Time Point After Shifted to Reoxygenation 18
3.5 Identification of NDRG1 and CCND3 from cDNA Microarray 19
3.6 CCND3 Silencing in MCF-7 20
3.7 Cell Cycle Regulation by CCND3 under Reoxygenation in MCF-7 20
3.8 Overexpression of NDRG1 in MCF-7 21
3.9 NDRG1 Suppressed Cell Migration Under Reoxygenation 22
3.10 Cell Migration Regulated by Oxidative Stress Through NDRG1 Expression 22
Chapter 4 Discussion 23
4.1 From gene expression changes to pathway regulation in MCF-7 during reoxygenation by genome-wide microarray analysis 23
4.2 Identification of candidate genes 26
4.3 The role of CCND3 in MCF-7 under reoxygenation 27
4.4 The role of NDRG1 under reoxygenation 28
Figures 32
Tables 41
Reference 43

List of Figures
Figure 1. Principle component analysis (PCA) of oxygen responsive genes in MCF7 cells during 24h of reoxygenation after hypoxia. 32
Figure 2. (A) Dynamics of differentially expressed genes during acclimatization to reoxygenation. 33
Figure 3. Enriched canonical pathways of genes responsive to reoxygenation. 34
Figure 4. Representative pathways of each time point after shifted to reoxygenation. 35
Figure 5. Quantitative real-time PCR validation of the top 10 genes 36
Figure 6. CCND3 expression levels were knockdowned by small RNA interference. 36
Figure 7. CCND3 silencing affected cell cycle phase distribution under reoxygenation. 37
Figure 8. Overexpression of NDRG1 in MCF-7. (A) Transcript levels of NDRG1 using RT-PCR. 38
Figure 9. Over-expression of NDRG1 reduced cell migration under reoxygenation. 39
Figure 10. NDRG1 expression level was reduced after ROS scavenger treatment under reoxygenation. 40


List of Tables
Table1. The top 10 genes with higher ranking in both gene network connectivity and gene expression ratio. 41
Table 2. Cell cycle analysis of CCND3 knock-down in MCF-7 under hypoxia or reoxygenation. 42

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