請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47851完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 阮雪芬(Hsueh-Fen Juan) | |
| dc.contributor.author | Chung-Cheng Hsu | en |
| dc.contributor.author | 許仲誠 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:22:26Z | - |
| dc.date.available | 2012-12-10 | |
| dc.date.copyright | 2010-08-16 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-10 | |
| dc.identifier.citation | 1. Lee RC, Feinbaum RL, & Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843-854.
2. Reinhart BJ, et al. (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403(6772):901-906. 3. Lee Y, et al. (2004) MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23(20):4051-4060. 4. Cai X, Hagedorn CH, & Cullen BR (2004) Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA 10(12):1957-1966. 5. Lee Y, et al. (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425(6956):415-419. 6. Bohnsack MT, Czaplinski K, & Gorlich D (2004) Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA 10(2):185-191. 7. Lund E, Guttinger S, Calado A, Dahlberg JE, & Kutay U (2004) Nuclear export of microRNA precursors. Science 303(5654):95-98. 8. Yi R, Qin Y, Macara IG, & Cullen BR (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17(24):3011-3016. 9. Hutvagner G, et al. (2001) A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293(5531):834-838. 10. Ketting RF, et al. (2001) Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15(20):2654-2659. 11. Song XM, Yan F, & Du LX (2006) [Components and assembly of RNA-induced silencing complex]. Yi Chuan 28(6):761-766. 12. Filip A (2007) [MiRNA--new mechanisms of gene expression control]. Postepy Biochem 53(4):413-419. 13. Mello CC & Conte D, Jr. (2004) Revealing the world of RNA interference. Nature 431(7006):338-342. 14. Fire A, et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806-811. 15. Ambros V, et al. (2003) A uniform system for microRNA annotation. RNA 9(3):277-279. 16. Kutter C & Svoboda P (2008) miRNA, siRNA, piRNA: Knowns of the unknown. RNA Biol 5(4):181-188. 17. Calin GA, et al. (2004) Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A 101(9):2999-3004. 18. Calin GA & Croce CM (2006) MicroRNAs and chromosomal abnormalities in cancer cells. Oncogene 25(46):6202-6210. 19. Esquela-Kerscher A & Slack FJ (2006) Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer 6(4):259-269. 20. Cho WC (2007) OncomiRs: the discovery and progress of microRNAs in cancers. Mol Cancer 6:60. 21. Zhu S, Si ML, Wu H, & Mo YY (2007) MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1). J Biol Chem 282(19):14328-14336. 22. Raval GN, et al. (2003) Loss of expression of tropomyosin-1, a novel class II tumor suppressor that induces anoikis, in primary breast tumors. Oncogene 22(40):6194-6203. 23. Johnson SM, et al. (2005) RAS is regulated by the let-7 microRNA family. Cell 120(5):635-647. 24. Szeberenyi J, Erhardt P, Cai H, & Cooper GM (1992) Role of Ras in signal transduction from the nerve growth factor receptor: relationship to protein kinase C, calcium and cyclic AMP. Oncogene 7(11):2105-2113. 25. Kometiani P, et al. (1998) Multiple signal transduction pathways link Na+/K+-ATPase to growth-related genes in cardiac myocytes. The roles of Ras and mitogen-activated protein kinases. J Biol Chem 273(24):15249-15256. 26. Ulku AS & Der CJ (2003) Ras signaling, deregulation of gene expression and oncogenesis. Cancer Treat Res 115:189-208. 27. Novara R, Coda R, Martone T, & Vineis P (1996) Exposure to aromatic amines and ras and c-erbB-2 overexpression in bladder cancer. J Occup Environ Med 38(4):390-393. 28. Akkiprik M, et al. (2008) Relationship between overexpression of ras p21 oncoprotein and K-ras codon 12 and 13 mutations in Turkish colorectal cancer patients. Turk J Gastroenterol 19(1):22-27. 29. Mudduluru G, et al. (2007) Loss of programmed cell death 4 expression marks adenoma-carcinoma transition, correlates inversely with phosphorylated protein kinase B, and is an independent prognostic factor in resected colorectal cancer. Cancer 110(8):1697-1707. 30. Mitchell PS, et al. (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 105(30):10513-10518. 31. Chang YM, et al. (2008) Prediction of human miRNAs using tissue-selective motifs in 3' UTRs. Proc Natl Acad Sci U S A 105(44):17061-17066. 32. Norbury C & Nurse P (1992) Animal cell cycles and their control. Annu Rev Biochem 61:441-470. 33. Park MT & Lee SJ (2003) Cell cycle and cancer. J Biochem Mol Biol 36(1):60-65. 34. Hartwell LH & Weinert TA (1989) Checkpoints: controls that ensure the order of cell cycle events. Science 246(4930):629-634. 35. Smith ML & Fornace AJ, Jr. (1996) Mammalian DNA damage-inducible genes associated with growth arrest and apoptosis. Mutat Res 340(2-3):109-124. 36. Weinberg RA (1995) The retinoblastoma protein and cell cycle control. Cell 81(3):323-330. 37. Paggi MG, Baldi A, Bonetto F, & Giordano A (1996) Retinoblastoma protein family in cell cycle and cancer: a review. J Cell Biochem 62(3):418-430. 38. Dyson N (1998) The regulation of E2F by pRB-family proteins. Genes Dev 12(15):2245-2262. 39. Bartek J, Bartkova J, & Lukas J (1996) The retinoblastoma protein pathway and the restriction point. Curr Opin Cell Biol 8(6):805-814. 40. Schena M, Shalon D, Davis RW, & Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270(5235):467-470. 41. Melle C, et al. (2003) Biomarker discovery and identification in laser microdissected head and neck squamous cell carcinoma with ProteinChip technology, two-dimensional gel electrophoresis, tandem mass spectrometry, and immunohistochemistry. Mol Cell Proteomics 2(7):443-452. 42. Chen HW, et al. (2005) Transcriptome analysis in blastocyst hatching by cDNA microarray. Hum Reprod 20(9):2492-2501. 43. Brooks G, Poolman RA, & Li JM (1998) Arresting developments in the cardiac myocyte cell cycle: role of cyclin-dependent kinase inhibitors. Cardiovasc Res 39(2):301-311. 44. Yu Q, et al. (2006) Requirement for CDK4 kinase function in breast cancer. Cancer Cell 9(1):23-32. 45. Elangovan S, Hsieh TC, & Wu JM (2008) Growth inhibition of human MDA-mB-231 breast cancer cells by delta-tocotrienol is associated with loss of cyclin D1/CDK4 expression and accompanying changes in the state of phosphorylation of the retinoblastoma tumor suppressor gene product. Anticancer Res 28(5A):2641-2647. 46. Shan W, Yang G, & Liu J (2009) The inflammatory network: bridging senescent stroma and epithelial tumorigenesis. Front Biosci 14:4044-4057. 47. Labib K & Diffley JF (2001) Is the MCM2-7 complex the eukaryotic DNA replication fork helicase? Curr Opin Genet Dev 11(1):64-70. 48. Bochman ML & Schwacha A (2008) The Mcm2-7 complex has in vitro helicase activity. Mol Cell 31(2):287-293. 49. Donovan S, Harwood J, Drury LS, & Diffley JF (1997) Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc Natl Acad Sci U S A 94(11):5611-5616. 50. Perkins G & Diffley JF (1998) Nucleotide-dependent prereplicative complex assembly by Cdc6p, a homolog of eukaryotic and prokaryotic clamp-loaders. Mol Cell 2(1):23-32. 51. Lutzmann M & Mechali M (2008) MCM9 binds Cdt1 and is required for the assembly of prereplication complexes. Mol Cell 31(2):190-200. 52. Masuda T, Mimura S, & Takisawa H (2003) CDK- and Cdc45-dependent priming of the MCM complex on chromatin during S-phase in Xenopus egg extracts: possible activation of MCM helicase by association with Cdc45. Genes Cells 8(2):145-161. 53. Zhu X, Ohtsubo M, Bohmer RM, Roberts JM, & Assoian RK (1996) Adhesion-dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2, and phosphorylation of the retinoblastoma protein. J Cell Biol 133(2):391-403. 54. Wolter F, Akoglu B, Clausnitzer A, & Stein J (2001) Downregulation of the cyclin D1/Cdk4 complex occurs during resveratrol-induced cell cycle arrest in colon cancer cell lines. J Nutr 131(8):2197-2203. 55. Mishra R & Das BR (2003) Early overexpression of Cdk4 and possible role of KRF and c-myc in chewing tobacco mediated oral cancer development. Mol Biol Rep 30(4):207-213. 56. Retzer-Lidl M, Schmid RM, & Schneider G (2007) Inhibition of CDK4 impairs proliferation of pancreatic cancer cells and sensitizes towards TRAIL-induced apoptosis via downregulation of survivin. Int J Cancer 121(1):66-75. 57. Willoughby JA, Sr., et al. (2009) Artemisinin blocks prostate cancer growth and cell cycle progression by disrupting Sp1 interactions with the cyclin-dependent kinase-4 (CDK4) promoter and inhibiting CDK4 gene expression. J Biol Chem 284(4):2203-2213. 58. Gatfield D, et al. (2009) Integration of microRNA miR-122 in hepatic circadian gene expression. Genes Dev 23(11):1313-1326. 59. Kai ZS & Pasquinelli AE (MicroRNA assassins: factors that regulate the disappearance of miRNAs. Nat Struct Mol Biol 17(1):5-10. 60. Lucas JJ, Szepesi A, Modiano JF, Domenico J, & Gelfand EW (1995) Regulation of synthesis and activity of the PLSTIRE protein (cyclin-dependent kinase 6 (cdk6)), a major cyclin D-associated cdk4 homologue in normal human T lymphocytes. J Immunol 154(12):6275-6284. 61. Sun F, et al. (2008) Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest. FEBS Lett 582(10):1564-1568. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47851 | - |
| dc.description.abstract | 微型核醣核酸microRNA（miRNA）是一群長度約20-23個鹼基之間的短片段內生型核酸，本身並不會轉譯出蛋白質，其功能和基因表現的調控有關，對於其目標基因能夠藉著與其訊息RNA (mRNA) 的3’-非轉譯區 (3’-UTR) 結合來進行負向調控，以抑制蛋白質的合成。最近的研究中發現微型核醣核酸除了與細胞的生長發育有密切關係之外，與癌症的發生與發展有密切的關係。此外，微型核醣核酸在細胞訊息傳遞路徑中所扮演的角色也逐漸被發現，但其確切的功能尚未完全解開。李文雄院士、施純傑博士與我們組成的團隊於2008年利用電腦預測的方法發現一個可能的微型核醣核酸p-27-5p，我們以人類乳癌細胞株MDA-MB-231及T-47D為材料進行研究，發現p-27-5p在這些細胞株中並不會有內生性的表現。以人工方式讓此兩株細胞表現p-27-5p後，在細胞生長曲線實驗中發現p-27-5p會導致細胞株T-47D生長減緩，而在細胞週期分析中發現p-27-5p會造成乳癌細胞株T-47D的細胞週期停滯，進一步用外顯子微陣列 (exon array) 及網路分析軟體Ingenuity Pathway Analysis分析p-27-5p對乳癌細胞株T-47D造成的基因調控網路影響，發現細胞表現p-27-5p後，細胞週期、細胞凋亡及癌症相關基因網路明顯受到改變。我們經由冷光酶活性測定分析驗證週期素依賴激酶第四型(cyclin-dependent kinase 4, CDK4) 為p-27-5p的目標基因，並利用西方墨點法證實p-27-5p會抑制T-47D細胞株CDK4的蛋白質表現以及RB1蛋白質之磷酸化程度，藉此抑制E2F1的活化，使細胞無法通過G1/S檢查點，進而抑制細胞增生。總而言之，本項研究發現以人工方法將p-27-5p送入乳癌細胞中，會抑制CDK4蛋白質的表現量，進而抑制其下游的基因調控網路，導致細胞週期受到影響，最終抑制癌細胞的生長。 | zh_TW |
| dc.description.abstract | MicroRNAs (miRNAs) are small, endogenous non-coding RNAs of 20–23 nucleotides in length that negatively regulate the expression of target genes at the post-transcriptional level. miRNAs have recently emerged as important regulators that play significant roles in tumorigenesis. The present study attempted to elucidate the functions of p-27-5p, a novel possible miRNA we recently discovered (Chang et al. 2008). In our study, we found that cell growth exhibited a decrease and showed that the breast cancer cell line T-47D were induced to arrest at G0/G1 phase after treatment with p-27-5p mimic. To understand the regulatory mechanism of p-27-5p, we performed exon array and Ingenuity Pathway Analysis to construct gene expression networks of p-27-5p-treated breast cancer cell line T-47D. The results demonstrated that several genes associated with cancer cell apoptosis or proliferation experienced an expression change after p-27-5p treatment. We demonstrated that cyclin-dependent kinase 4 (CDK4) is the target of p-27-5p by using luciferase reporter assay. Previous studies indicated that CDK4 can stimulate cell cycle progression and is often overexpressed in cancer cells. We also demonstrated that p-27-5p downregulates CDK4 protein expression and RB1 phosphorylation by using western blotting. Taken together, our data suggest this artificial miRNA, p-27-5p, plays an important role in cancer progression and has the potential to be applied in breast cancer therapy. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:22:26Z (GMT). No. of bitstreams: 1 ntu-99-R97b43021-1.pdf: 3645688 bytes, checksum: d192db59d425ac8f6c30cd26ebc5f36c (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 口試委員審定書 i
致謝 ii 中文摘要 iv Abstract v 縮寫表 vi 第一章 前言與背景介紹 1 1-1 微型核醣核酸 (miRNAs) 的介紹 1 1-2 微型核醣核酸 (miRNA) 和小型干擾核醣核酸 (siRNA) 的差異 2 1-3 微型核醣核酸在癌症上的角色 2 1-4 p-27-5p的發現 4 1-5 細胞週期 (cell cycle) 的介紹 4 1-6 外顯子微陣列 (exon array) 介紹 5 1-7 IPA (Ingenuity Pathway Analysis) 的介紹 6 1-8 研究目標 6 第二章 實驗材料與方法 7 2-1 細胞培養 7 2-1-1 解凍增殖 8 2-1-2 繼代培養 8 2-1-3 冷凍長期保存 8 2-1-4 細胞計數 9 2-2 細胞總核糖核酸 (含微型核糖核酸) 萃取 9 2-3 反轉錄反應 (Reverse transcription) 9 2-4 即時定量聚合酶連鎖反應 (Real-time PCR) 10 2-5 轉染作用 (Transfection) 10 2-6 以Roche xCELLigence System觀察細胞生長情形 10 2-7 外顯子微陣列 11 2-8 基因表現資料統計分析 12 2-9 基因表現網路分析 12 2-10 pMIR-REPORT-CDK4 3’UTR expression vector質體構築 13 2-10-1 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 13 2-10-2 1% DNA洋菜膠體置備和電泳分析 13 2-10-3 DNA限制酵素反應 14 2-10-4 膠體萃取DNA (bioman EasyPure PCR/Gel Extraction kit) 14 2-10-5 接合作用和轉型作用 14 2-10-6 菌落PCR篩選和定序 15 2-11 冷光酶活性測定分析 (Luciferase reporter assay) 15 2-11-1 轉染作用 15 2-11-2 Luciferase與β-galactosidase訊號偵測 16 2-12 西方墨點法 (Western blot) 16 2-13 以流式細胞儀進行細胞週期分析 (cell cycle analysis) 17 2-14 以流式細胞儀進行細胞凋亡分析 (cell apoptosis analysis) 18 第三章 結果 19 3-1. 利用real-time PCR測定p-27-5p在正常乳腺細胞株MCF-10A及乳癌細胞株T-47D及MDA-MB-231中的內生性表現量 19 3-2. 利用real-time PCR驗證p-27-5p在轉染後之表現量 19 3-3. 利用Roche xCELLigence DP system觀察正常乳腺細胞株及乳癌細胞轉染p-27-5p後之生長曲線 20 3-4. 利用流式細胞儀分析p-27-5p對細胞週期之影響 21 3-5. 利用流式細胞儀分析p-27-5p是否造成細胞凋亡 21 3-6. 利用Partek Genomics Suite軟體將外顯子微陣列資料做初步統計分析 22 3-7. 利用Ingenuity Pathway Analysis軟體分析基因表現資料 23 3-8. 利用real-time PCR驗證CDK4在轉染p-27-5p後之表現量 23 3-9. 利用NCBI blast工具比對CDK4 3’-UTR與p-27-5p 24 3-10. 利用冷光酶活性測定分析驗證p-27-5p能調控其目標基因CDK4 24 3-11. 利用西方墨點法驗證在T-47D細胞株中p-27-5p能造成其目標基因CDK4的蛋白質表現量下降且在MCF-10A細胞株中無此現象 25 3-12. 利用西方墨點法來驗證p-27-5p對於RB1的磷酸化是否造成影響 25 第四章 討論 26 圖 31 表 50 參考文獻 56 附錄 63 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討人工微型核醣核酸p-27-5p於乳癌細胞株T-47D基因調控網路之影響 | zh_TW |
| dc.title | Gene Regulatory Networks of Artificial MicroRNA p-27-5p-Treated Human Breast Cancer Cell Line T-47D | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃宣誠(Hsuan-Cheng Huang),李文雄(Wen-Hsiung Li),施純傑(Arthur Chun-Chieh Shih),陳水田(Shui-Tein Chen) | |
| dc.subject.keyword | 微型核醣核酸,乳癌,細胞週期,外顯子微陣列,基因調控網路, | zh_TW |
| dc.subject.keyword | miRNAs,breast cancer,cell cycle,exon array,regulatory network, | en |
| dc.relation.page | 73 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-10 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-99-1.pdf 目前未授權公開取用 | 3.56 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。