請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77965完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 胡忠怡 | zh_TW |
| dc.contributor.author | 李文博 | zh_TW |
| dc.contributor.author | Man-Pok Lei | en |
| dc.date.accessioned | 2021-07-11T14:38:28Z | - |
| dc.date.available | 2024-03-21 | - |
| dc.date.copyright | 2017-09-12 | - |
| dc.date.issued | 2017 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | References
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Oncol Rep, 2013. 30(4): p. 1631-8. 98. Fisher, K.W., B. Das, H.S. Kim, B.K. Clymer, D. Gehring, D.R. Smith, D.L. Costanzo-Garvey, M.R. Fernandez, M.G. Brattain, D.L. Kelly, J. MacMillan, M.A. White, and R.E. Lewis, AMPK Promotes Aberrant PGC1beta Expression To Support Human Colon Tumor Cell Survival. Mol Cell Biol, 2015. 35(22): p. 3866-79. 99. Mihaylova, M.M. and R.J. Shaw, The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol, 2011. 13(9): p. 1016-23. 100. Sahin, E. and R.A. DePinho, Axis of ageing: telomeres, p53 and mitochondria. Nat Rev Mol Cell Biol, 2012. 13(6): p. 397-404. 101. Shackelford, D.B. and R.J. Shaw, The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat Rev Cancer, 2009. 9(8): p. 563-75. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77965 | - |
| dc.description.abstract | 神經母細胞瘤(Neuroblastoma, NB)為兒童癌症中最常見之顱外固態腫瘤，其佔兒童癌症死亡率約12%，而發生轉錄因子MYCN增幅的NB病人存活率較差。微核醣核酸(microRNA or miRNA)為一種非轉譯之小核醣核酸分子，透過轉錄後機制抑制標的基因的表現。過往miRNA研究中發現MYCN增幅的NB腫瘤與MYCN轉殖小鼠中皆有一群miRNA表現量顯著下降，包括miR-X。已知miR-X在非小細胞肺癌、腎臟惡性腫瘤與神經纖維肉瘤中表現量顯著減低，並具抑癌作用。初步分析腫瘤中miR-X表現量較高的NB病人，其存活率顯著較好。顯示miR-X在NB中可能有抑癌作用。然而其確實調控角色則尚不清楚。
分析台大兒童醫院40位NB病人於進行化療前收集的腫瘤檢體內miR-X表現量，發現臨床分期上風險較高的病人miR-X表現量顯著較低，而miR-X表現量高的病人的整體存活率與無事故存活率都較高。在六株人類NB細胞株中，有MYCN增幅的細胞株miR-X表現量有較低之趨勢。在有MYCN增幅的兩株NB細胞(SK-N-DZ，SK-N-BE)中以用慢病毒轉導使過度表現miR-X後細胞生長速度明顯減緩，分析細胞週期時發現有G0/G1停滯的現象；細胞非貼附生長能力、侵襲能力與遷移能力也顯著下降。顯示miR-X有抑制NB細胞腫瘤惡性程度的功能。分析過度表現miR-X後細胞的ATP產量與葡萄糖吸收量都有顯著上升，說明miR-X可調控細胞之糖類代謝。以微陣列(microarray)分析發現共151個基因在過表現miR-X後基因表現量顯著下降，其中15個基因為可能直接受miR-X調控表現之分子，我們鎖定在R2網路基因資料庫分析中顯示與NB臨床預後有關的PPARGC1B作後續研究。 PPARGC1B為一參與粒腺體新生(mitochondrial biogenesis)的基因，同時亦可調控細胞能量代謝。已知PPARGC1B在大腸癌及乳癌中扮演致癌角色，腫瘤細胞中PPARGC1B高表現已證實與增進癌細胞非貼附生長能力、降低細胞ATP產量生成相關。而進一步以RT-qPCR方法發現過度表現miR-X的兩NB細胞株，其PPARGC1B表現量確實下降，與微陣列分析結果符合。在人類NB細胞株中，具有MYCN增幅之SK-N-DZ與SK-N-BE細胞中PPARGC1B表現量較高，我們利用shRNA將SK-N-DZ細胞中PPARGC1B基因默化。而SK-N-DZ細胞在默化PPARGC1B後生長速度顯著下降，與過表現miR-X後的結果相符。 未來我們將通過reporter assay分析miR-X是否確實能直接調降PPARGC1B基因表現，同時探討PPARGC1B通過何種影響何種下游分子從而抑制NB細胞腫瘤惡性度；另外亦會建立miR-X剔除/默化的NB細胞株，從而確實其在NB中扮演抑癌角色的假設。 | zh_TW |
| dc.description.abstract | Neuroblastoma (NB) is an extracranial solid tumor with aggressive and metastatic features affecting young children. Patients with MYCN proto-oncogene amplification will have poor prognosis and survival. MicroRNA belongs to the short non-coding RNA family known to post-transcriptionally regulate the expression of their target genes, and play important roles in tumorigenesis. Recent miRNA profiling studies revealed a group of miRNAs were down-regulated in MYCN-amplified (MNA) NB, including miR-X, which was known as a tumor suppressor in non-small cell lung cancer and renal cell carcinoma. This suggests a tumor suppressor role of miR-X in NB.
Here we evaluated tumor miR-X expression in 40 primary NB tumors obtained from untreated patients, it was noted that patients with INSS low risk stages (stages 1, 2) or without MNA had a higher miR-X expression in the tumors. Patients with higher tumor miR-X expression had better survival. Human NB cell lines bearing MYCN amplification (SK-N-DZ and SK-N-BE) showed a relatively lower miR-X expression as compared to cells without MNA (SK-N-SH, SH-SY-5Y). Overexpression of miR-X in MNA-NB cells by lentiviral transduction downregulated cell proliferation, anchorage-independent colony formation, migration ability and matrigel invasiveness. Furthermore, overexpression of miR-X increased glucose uptake and ATP production in MNA NB cell lines, indicating its effect on regulating energy metabolism. Microarray analysis identified 151 genes were significantly downregulated in miR-X-overexpressed compared to the control SK-N-DZ cells. Of particular interest, PPARGC1B, which encodes PGC-1 that is involved in mitochondria biogenesis and regulating glucose metabolism, and has shown significant clinical impact in the public NB database R2, was found to be decreased in the miR-X-overexpressed cells. We then generated PPARGC1B knock down SK-N-DZ cell using shRNA. Knocking down PPARGC1B significantly inhibited SK-N-DZ cell growth and anchorage-independent clonogenic formation. We will further investigate whether miR-X can inhibit PPARGC1B expression through direct targeting to its 3’-UTR using reporter assay. Secondary, the possible PPARGC1B down-stream targets will be analyzed using Western blot and RT-qPCR, to clarify the miR-X-PPARGC1B and its down-stream regulating pathway that is responsible to the phenotypic changes in the miR-X overexpressed NB cells. MiR-X knock out/down assay will be also necessary for validation of the tumor suppressive effects of miR-X in NB cell lines. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:38:28Z (GMT). No. of bitstreams: 1 ntu-106-R04424016-1.pdf: 3590568 bytes, checksum: de754b527568f757a2548431df582a25 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 口試委員審定書 i
誌謝 ii 摘要 iii ABSTRACT v 目錄 vii 圖表目錄 xi 附錄目錄 xii 第一章 緒論 1 1.1 神經母細胞瘤 1 1.1.1 神經母細胞瘤簡介 1 1.1.2 神經母細胞瘤致病機轉 1 1.1.3 神經母細胞瘤臨床表現 2 1.1.4 神經母細胞瘤臨床疾病診斷與分期 3 1.1.5 神經母細胞病理特徵及分類 3 1.1.6 神經母細胞分子標記及預後指標 4 1.1.7 神經母細胞治療策略 6 1.2 微核醣核酸 7 1.2.1 微核醣核酸簡介 7 1.2.2 微核醣核酸生理功能及生理意義 7 1.2.3 微核醣核酸在癌症中扮演的角色 8 1.2.4 微核醣核酸-30d 9 1.3 瓦氏效應 10 1.4 研究假說 11 第二章 研究目的與實驗設計 12 2.1 研究目的 12 2.2 實驗設計 12 第三章 材料與方法 13 3.1 實驗材料 13 3.1.1 細胞株 13 3.1.2 臨床檢體 13 3.1.3 試劑試藥、抗體清單 13 3.1.4 試劑套組 15 3.1.5 溶液試劑配方 16 3.1.6 實驗儀器 18 3.1.7 質體 19 3.1.8 載體 19 3.1.9 miR-30d cloning及RT-qPCR引子序列 20 3.1.10 軟體與網路工具 20 3.2 實驗方法 22 3.2.1 建立pLKO.1-30d質體 22 3.2.2 慢病毒製備 23 3.2.3 解凍細胞、繼代培養 23 3.2.4 慢病毒轉導 24 3.2.5 細胞增生及活性分析 24 3.2.6 非貼附生長能力分析 24 3.2.7 細胞遷移試驗 25 3.2.8 細胞侵襲能力試驗 25 3.2.9 細胞週期分析 25 3.2.10 核醣核酸/微核醣核酸相對表現量分析 26 3.2.11 三磷酸腺苷產量試驗 28 3.2.12 葡萄糖吸收試驗 29 3.2.13 基因微陣列分析 29 3.2.14 西方墨點法 29 3.2.15 數據統計分析 31 第四章 實驗結果 32 4.1 神經母細胞瘤腫瘤中miR-30d表現量分析 32 4.1.1 miR-30d低表現量與疾病不良分群相關 32 4.1.2 miR-30d低表現量與病人不良預後相關 32 4.2 神經母細胞瘤細胞株miR-30d表現量 32 4.3 於NB細胞株中過度miR-30d後影響細胞表徵 33 4.3.1 過表現miR-30d於SK-N-DZ、SK-N-BE 33 4.3.2 miR-30d抑制細胞生長 33 4.3.3 miR-30d抑制細胞腫瘤惡性度 33 4.4 過表現miR-30d影響細胞糖類代謝 34 4.4.1 miR-30d提高細胞ATP產量 34 4.4.2 miR-30d提高細胞葡萄糖吸取量 34 4.4.3 過表現miR-30d後細胞糖類代謝相關基因表現量 35 4.5 PPARGC1B為具研究潛力之miR-30d目標基因 35 4.5.1 cDNA微陣列分析 35 4.5.2 選擇PPARGC1B為後續研究對象 36 4.5.3 過表現miR-30d後PPARGC1B表現量下降 36 4.6 於SK-N-DZ中默化PPARGC1B後影響細胞表徵 37 4.6.1 NB細胞株中PPARGC1B表現量 37 4.6.2 於SK-N-DZ中默化PPARGC1B 37 4.6.3 默化PPARGC1B抑制SK-N-DZ細胞生長 37 4.6.4 默化PPARGC1B抑制SK-N-DZ細胞非貼附生長能力 38 第五章 討論 39 參考文獻 44 圖與表 54 附錄 76 | - |
| dc.language.iso | zh_TW | - |
| dc.title | 微核醣核酸-X在神經母細胞瘤中之抑癌角色 | zh_TW |
| dc.title | Tumor suppressive effects of a microRNA-X in human neuroblastoam | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 105-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 周獻堂;許文明;林亮音;郭遠燁 | zh_TW |
| dc.contributor.oralexamcommittee | ;;; | en |
| dc.subject.keyword | 神經母細胞瘤,微核醣核酸,抑癌因子,PPARGC1B, | zh_TW |
| dc.subject.keyword | neuroblastoma,microRNA,tumor suppressor,PPARGC1B, | en |
| dc.relation.page | 90 | - |
| dc.identifier.doi | 10.6342/NTU201701838 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2017-07-25 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | - |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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