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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 俞松良(Sung-Liang Yu) | |
dc.contributor.author | Ching-Cheng Chiang | en |
dc.contributor.author | 蔣景程 | zh_TW |
dc.date.accessioned | 2021-06-07T23:42:21Z | - |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-07-25 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16624 | - |
dc.description.abstract | 肺癌是世界各地研究中死亡人數最多的癌症。而轉移與否是決定病人預後與存活率的最重要的指標之一。為了研究肺癌的轉移機制,實驗室先前成功地建立了一系列具有不同侵襲能力的肺腺癌細胞株。最近,我們利用微陣列生物晶片分析找出一些可能與癌症侵襲能力調控相關的基因。因此,本研究著重研究其中兩個抑癌基因:小眼畸形相關轉錄因子及甲硫腺苷磷酸化酶,並探討這兩個基因抗轉移的調控機制。首先,研究結果指出,在高侵襲能力的細胞CL1-5中,小眼畸形相關轉錄因子其mRNA以及蛋白質表現量均顯著減少。在臨床結果分析中,小眼畸形相關轉錄因子與肺癌病人的總存活期與無病存活期均呈現顯著負相關。而為了研究小眼畸形相關轉錄因子對於癌細胞轉移能力的影響,我們利用RNA干擾技術處理低侵襲能力的細胞株,CL1-0,抑制其MITF表現。結果證明此基因被抑制後,可以在活體外促進癌細胞的移動、侵襲以及生長,還可以在活體內促進腫瘤的生長及轉移。進一步利用生物晶片及生物途徑分析,推測該基因的調控是透過WNT/ Frizzled傳導路徑。而另一部分,甲硫腺苷磷酸化酶,在CL1-5中,其DNA拷貝數、mRNA 以及蛋白質表現量也均顯著減少。而在CL1-5中將甲硫腺苷磷酸化酶過量表現,不但可以在活體外抑制癌細胞株的移動、侵襲能力以及腫瘤形成,還可在活體內減少腫瘤轉移及生長。此外,甲硫腺苷磷酸化酶的表現和臨床病人存活結果也呈現顯著負相關。在調控機制上,將甲硫腺苷磷酸化酶的酵素活性中心突變,會使得其失去抑制轉移的能力。這表示甲硫腺苷磷酸化酶需要其本身的酵素功能,以及對於細胞能量ATP的調節,來執行其在肺癌中所具有的抑癌能力。最後,經由生物晶片分析發現,轉錄因子 STAT1,在甲硫腺苷磷酸化酶所影響的腫瘤侵襲及轉移能力中,有可能部分參與到其中的調控。總結以上結果,我們可以證實小眼畸形相關轉錄因子及甲硫腺苷磷酸化酶為抑癌基因,並且本研究可提供在肺癌診斷及治療上新的思維。 | zh_TW |
dc.description.abstract | Lung cancer is the leading cause of cancer death worldwide. Metastasis is one of the most important characteristics to determine outcome and survival of cancer patients. To feasibly investigate the mechanism and regulation of lung cancer metastasis, a series of lung adenocarcinoma cell lines with varying degrees of invasiveness was established before. Recently, by using expression microarrays and the cell line model, we identified a panel of invasiveness-associated genes. Here, I mainly investigate the underlying anti-metastasis mechanisms of two new tumor-suppressor genes, MITF and MTAP, in lung cancer. First, the mRNA and protein levels of MITF were dramatically reduced in the highly invasive cells, CL1-5, and MITF expreesion was negatively correlated with overall and disease-free survivals of lung cancers. To investigate the effect of MITF on metastasis, the lower invasive CL1-0 cells were treated by the RNAi specific against MITF. The results revealed that the reduction of MITF promotes tumor cell migration, invasion and anchorage-independent growth in vitro and also increases metastasis and tumorigenesis in vivo. The anti-metastasis activity of MITF is through WNT/ Frizzled signaling pathway identified by expression microarray and pathway analysis. In the study of MTAP, we found that the gene copy number, mRNA and protein levels of MTAP are lower in CL1-5 cells. Overexpression of MTAP in CL1-5 significantly inhibited cell migration, invasion and tumorigenesis in vitro, and decreased tumor metastasis and growth in vivo. Moreover, MTAP exepression is negatively correlated with clinical outcome of NSCLC patients. The mutation of MTAP catalytic site totally eliminated the anti-metastatic activities. The fact indicated that the MTAP enzymatic activity and the ATP production are necessary for the tumor suppressive activity of MTAP in lung cancer. And the transcription factor, STAT1, might be involved at leat partially in the MTAP-mediated tumor suppression identified by microarray analysis. Our results reveal that MITF and MTAP act as the tumor suppressors and provide a new insight for cancer prognosis and therapy. | en |
dc.description.provenance | Made available in DSpace on 2021-06-07T23:42:21Z (GMT). No. of bitstreams: 1 ntu-103-D96424004-1.pdf: 19947090 bytes, checksum: 98d4694e61926a18560c3bfbaae651e1 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | Acknowledge i
中文摘要 vi Abstract vii Contents ix List of Figures xii List of Tables xv Chapter I: Suppression of Microphthalmia-associated Transcription Factor Enhances Tumor Malignancy in Lung Cancer 1 A. Abstract 2 B. Introduction 4 1. Lung cancer 6 2. Histological classification of lung cancer 8 3. Tumor migration, invasion and metastasis 11 4. Waardenburg syndrome 14 5. Genetic background of Microphthalmia-associated transcription factor 15 6. The special isoform of MITF: MITF-M 15 7. The biological regulation of MITF in melanoma 16 8. Genetic alteration in lung cancer 18 C. Materials and Methods 24 1. Cell culture 24 2. Isoform-specific RT-PCR 24 3. Western blot analysis 25 4. shRNA stable transfection and construction of expression vector 26 5. Cell proliferation and anchorage independent colony formation 27 6. Patients and tissue specimens 28 7. Quantitative real-time RT-PCR 28 8. Identification of MITF binding sites in regulatory element 29 9. Chromatin immunoprecipitation assay (ChIP) 30 10. cDNA microarray system 31 11. Oligonucleotide microarray analysis 32 12. Statistical analysis 33 13. Migration and matrigel invasion assays 34 14. Matrigel plug assay and tumorigenicity in SCID 35 15. Quantitative detection of human tumor cell metastasis in vivo 36 D. Results 38 1. MITF Expression Predicts Clinical Outcome in Patients with NSCLC 38 2. Silencing MITF Enhances NSCLC Cell Migration and Invasion Abilities 40 3. Silencing MITF also Promotes cells tumorigenesis but not in cells proliferation 41 4. Knock-down MITF Expression Promotes Tumor Growth and Metastasis, but Suppresses Angiogenesis In Vivo 42 5. The Possible Downstream Gene, FZD7, Which Binds Directly with MITF, Regulates Cell Migration/Invasion Ability 43 E. Discussions 45 Chapter II: Methylthioadenosine Phosphorylase Inhibits Tumor Invasion and Metastasis via Energy Regulation 52 A. Abstract 53 B. Introduction 55 1. Methylthioadenosine 58 2. The function of methylthioadenosine phosphorylase 59 3. The role of MTAP in tumor cells 60 C. Materials and Methods 62 1. Cell culture 62 2. Oligonucleotide microarray analysis 62 3. Construction of expression vector and stable transfection 63 4. Small-interfering RNA transient transfection 64 5. Western blot analysis. 65 6. Migration and matrigel invasion assays 66 7. Cell proliferation and colony formation assays 66 8. Wound healing assay 67 9. Immunofluorescence 68 10. Angiogenesis 69 11. Anchorage independent colony formation 69 12. Real-Time quantitative RT-PCR 70 13. In vivo metastasis 71 14. Patients and tissue specimens 71 15. Statistical analysis 72 16. Cellular ATP content measurement 73 17. Measurements of oxygen consumption rate (OCR) 73 18. In vivo tumorigenicity in SCID 74 19. Array CGH 74 D. Results 76 1. MTAP is a potential tumor-suppressor gene in lung adenocarcinomas 76 2. Up-regulation of MTAP results in CL1-5 cell mobility lost 77 3. MTAP also change invasion ability in other lung cancer cell line 79 4. MTAP also promotes cells tumorigenesis, but not involved in cell proliferation and angiogenesis 80 5. MTAP reduces lung cancer cell metastasis in vivo 81 6. MTAP also reduces lung cancer cell tumorgenesis in vivo 82 7. MTAP expression were correlated with the NSCLC survival 82 8. Identification of MTAP downstream genes by microarray analysis 83 9. MTAP expression and suppression of cell movement and cytoskeleton remodeling 84 10. MTAP regulates cell invasion through a mechanism dependent its intrinsic phosphorylase activity 84 11. MTAP inhibited cell invasion through reducing cellular ATP production 86 12. Cancer cells polyamine pathway was influenced by MTAP induction 88 13. MTAP might promote the adenosine deaminase gene expression through signal transducers and activators of transcription 1 (STAT1) up-regulation 90 E. Discussion 91 Figures 96 Tables 160 References 171 Appendices 193 | |
dc.language.iso | en | |
dc.title | 小眼畸形相關轉錄因子及甲硫腺苷磷酸化酶在肺癌轉移角色之研究 | zh_TW |
dc.title | The role of Microphthalmia-associated Transcription Factor (MITF) and Methylthioadenosine Phosphorylase (MTAP) on metastasis in lung cancer | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 陳健尉(Jeremy J.W Chen),陳惠文(Huei-Wen Chen),張正琪(Cheng-Chi Chang),郭錦樺(Ching-Hua Kuo),蘇剛毅(Kang-Yi Su) | |
dc.subject.keyword | 肺癌,癌轉移,抑癌基因,小眼畸形相關轉錄因子,甲硫腺?磷酸化?, | zh_TW |
dc.subject.keyword | lung cancer,metastasis,tumor suppressors,MITF,MTAP, | en |
dc.relation.page | 204 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2014-07-28 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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