跳到主要內容

臺灣博碩士論文加值系統

(3.95.131.146) 您好!臺灣時間:2021/07/29 02:43
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:黃珮珍
研究生(外文):Peu-Chen Huang
論文名稱:建立可多次取樣追蹤microRNA活性之報導系統
論文名稱(外文):Establishment of a Multi-sampling Reporter System to Monitor Activities of microRNAs
指導教授:歐樂君
指導教授(外文):Lo-Chun Au
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:59
中文關鍵詞:播報系統重複取樣分泌型酵素microRNA活性培養液
外文關鍵詞:reporter systemmulti-samplingsecretable enzymesmiRNA activitymedium
相關次數:
  • 被引用被引用:0
  • 點閱點閱:189
  • 評分評分:
  • 下載下載:48
  • 收藏至我的研究室書目清單書目收藏:0
目前有許多方法可用於定量miRNA或偵測miRNA的活性。而北方墨點法(Northern blot)、即時定量RT-PCR、生物晶片(microarray)和螢火蟲螢光酵素播報系統(firefly luciferase reporter system)則是最廣為使用的幾種方法。然而這些方法都需要將待測細胞溶解後方可進行分析,因此無法持續監控同一群細胞在不同時間點的miRNA表現。在以上述方法進行不同時間點的miRNA偵測時必須要培養許多盤細胞,而此舉將增將實驗的複雜性,且細胞盤與盤間的差異可能會對實驗的精確度造成影響。有鑑於此,在本實驗中我們利用兩種可由細胞分泌出來的生物冷光酵素,一個用於偵測miRNA而另一個則用於監控轉染的效率,成功的建立一個可重複取樣的播報系統。由於該系統只需收取少量的細胞培養液即可進行分析,因此能在長時間中對同一群細胞進行重複取樣並偵測該細胞在不同時間點miRNA的活性。而在本實驗中我們也對該系統的特性、可行性與優點進行深入的探討。
There are several methods to detect the relative levels or activities of miRNAs including Northern blot analysis, quantitative real-time RT-PCR, microarray and firefly luciferase reporter system. However, lysis of tested cells is necessary for all of these methods. Therefore, we cannot use these methods to continuously monitor a miRNA in the same population of cells, and need to prepare several sets of wells/dishes of cells for tracking the alteration of a miRNA level in the time-course studies. It is uneconomical and experimental accuracy may be compromised by the variations between samples.
In this study, we developed a multi-sampling reporter system by using two secretable bioluminescence-generating enzymes ─ Metrida luciferase and secreted alkaline phosphatase. One is for a reporter and the other for an internal control. Therefore we can monitor miRNA activities in the same population of cells over time simply by withdrawing aliquots of the culture medium followed by assays. We also demonstrated characteristic, practicability and benefits of this system are in this report.
論文電子檔著作權授權書 i
論文審定同意書 ii
誌謝 iii
中文摘要 iv
Abstract v
Table of Contents vi
List of Figures viii
Chapter 1: Introduction 1
1.1 An overview of microRNAs 1
1.2 Quantification of miRNAs 3
1.3 The secretable reporting enzymes 6
1.4 miR-15/16 family 8
1.5 Objectives 10
Chapter 2: Materials and Methods 12
2.1 Cell lines and cultures 12
2.2 The plasmids for the multi-sampling reporter system 12
2.3 Assay of MLuc activity 14
2.4 Assay of SEAP activity 15
2.5 Transfection of HEK293T 16
2.6 Cell lysis 16
2.7 Detection of secretable MLuc by X-ray films 16
2.8 Detection of level change of endogenous miR-16 17
2.9 Statistical analysis 18
Chapter 3: Results 19
3.1 The plasmids for multi-sampling reporter system 19
A. Screening of pMLuc-UTR by PCR 19
B. Sequencing result of pMLuc-UTR 20
3.2 Characterization of the multi-sampling reporter system 20
A. Detection of secretable MLuc by X-ray films 21
B. Detection of the reporting enzymes in media and lysates 21
C. Comparison between variations in commercial assays and modified assays 21
D. Comparison with the results made with different reagents 22
E. Background signals of MLuc and SEAP in culture media 23
F. Stability of MLuc and SEAP after freeze 23
G. Stability of MLuc and SEAP in culture media 23
H. Effects of dilution on repression folds 23
3.3 Reporting ability of the multi-sampling reporter system 24
A. Responsibility of the multi-sampling reporter system to pre-miR-16 24
B. Reporting ability of multi-sampling 24
C. Sensitivity of the multi-sampling reporting system 25
D. Detection of level change of endogenous miR-16 26
Chapter 4: Conclusion and Discussion 27
References 45
Appendix 48
[1] V. Ambros, The functions of animal microRNAs. Nature 431 (2004) 350-5.
[2] D.P. Bartel, MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116 (2004) 281-97.
[3] I. Bentwich, A. Avniel, Y. Karov, R. Aharonov, S. Gilad, O. Barad, A. Barzilai, P. Einat, U. Einav, E. Meiri, E. Sharon, Y. Spector, and Z. Bentwich, Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet 37 (2005) 766-70.
[4] K.C. Miranda, T. Huynh, Y. Tay, Y.S. Ang, W.L. Tam, A.M. Thomson, B. Lim, and I. Rigoutsos, A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell 126 (2006) 1203-17.
[5] A. Rodriguez, S. Griffiths-Jones, J.L. Ashurst, and A. Bradley, Identification of mammalian microRNA host genes and transcription units. Genome Res 14 (2004) 1902-10.
[6] S. Baskerville, and D.P. Bartel, Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA 11 (2005) 241-7.
[7] Y. Lee, C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee, P. Provost, O. Radmark, S. Kim, and V.N. Kim, The nuclear RNase III Drosha initiates microRNA processing. Nature 425 (2003) 415-9.
[8] R. Yi, Y. Qin, I.G. Macara, and B.R. Cullen, Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17 (2003) 3011-6.
[9] A. Esquela-Kerscher, and F.J. Slack, Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer 6 (2006) 259-69.
[10] M.A. Valencia-Sanchez, J. Liu, G.J. Hannon, and R. Parker, Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev 20 (2006) 515-24.
[11] W. Filipowicz, S.N. Bhattacharyya, and N. Sonenberg, Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9 (2008) 102-14.
[12] Y.W. Kong, I.G. Cannell, C.H. de Moor, K. Hill, P.G. Garside, T.L. Hamilton, H.A. Meijer, H.C. Dobbyn, M. Stoneley, K.A. Spriggs, A.E. Willis, and M. Bushell, The mechanism of micro-RNA-mediated translation repression is determined by the promoter of the target gene. Proc Natl Acad Sci U S A 105 (2008) 8866-71.
[13] R.C. Lee, R.L. Feinbaum, and V. Ambros, The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75 (1993) 843-54.
[14] C.Z. Chen, L. Li, H.F. Lodish, and D.P. Bartel, MicroRNAs modulate hematopoietic lineage differentiation. Science 303 (2004) 83-6.
[15] Y. Zhao, E. Samal, and D. Srivastava, Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature 436 (2005) 214-20.
[16] J. Takamizawa, H. Konishi, K. Yanagisawa, S. Tomida, H. Osada, H. Endoh, T. Harano, Y. Yatabe, M. Nagino, Y. Nimura, T. Mitsudomi, and T. Takahashi, Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res 64 (2004) 3753-6.
[17] P. Xu, S.Y. Vernooy, M. Guo, and B.A. Hay, The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism. Curr Biol 13 (2003) 790-5.
[18] M.Z. Michael, O.C. SM, N.G. van Holst Pellekaan, G.P. Young, and R.J. James, Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol Cancer Res 1 (2003) 882-91.
[19] J.A. Chan, A.M. Krichevsky, and K.S. Kosik, MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res 65 (2005) 6029-33.
[20] K.A. Cissell, and S.K. Deo, Trends in microRNA detection. Anal Bioanal Chem (2009).
[21] J.Y. Lee, S. Kim, W. Hwang do, J.M. Jeong, J.K. Chung, M.C. Lee, and D.S. Lee, Development of a dual-luciferase reporter system for in vivo visualization of MicroRNA biogenesis and posttranscriptional regulation. J Nucl Med 49 (2008) 285-94.
[22] E.A. Miska, E. Alvarez-Saavedra, M. Townsend, A. Yoshii, N. Sestan, P. Rakic, M. Constantine-Paton, and H.R. Horvitz, Microarray analysis of microRNA expression in the developing mammalian brain. Genome Biol 5 (2004) R68.
[23] W.P. Tsang, and T.T. Kwok, Epigallocatechin gallate up-regulation of miR-16 and induction of apoptosis in human cancer cells. J Nutr Biochem (2009).
[24] Z. Liu, M. Winters, M. Holodniy, and H. Dai, siRNA delivery into human T cells and primary cells with carbon-nanotube transporters. Angew Chem Int Ed Engl 46 (2007) 2023-7.
[25] R. Shi, and V.L. Chiang, Facile means for quantifying microRNA expression by real-time PCR. Biotechniques 39 (2005) 519-25.
[26] G.A. Calin, and C.M. Croce, MicroRNA signatures in human cancers. Nat Rev Cancer 6 (2006) 857-66.
[27] V. Vopalensky, T. Masek, O. Horvath, B. Vicenova, M. Mokrejs, and M. Pospisek, Firefly luciferase gene contains a cryptic promoter. RNA 14 (2008) 1720-9.
[28] E.M. Thompson, S. Nagata, and F.I. Tsuji, Cloning and expression of cDNA for the luciferase from the marine ostracod Vargula hilgendorfii. Proc Natl Acad Sci U S A 86 (1989) 6567-71.
[29] Y. Nakajima, K. Kobayashi, K. Yamagishi, T. Enomoto, and Y. Ohmiya, cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca. Biosci Biotechnol Biochem 68 (2004) 565-70.
[30] S.V. Markova, S. Golz, L.A. Frank, B. Kalthof, and E.S. Vysotski, Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme. J Biol Chem 279 (2004) 3212-7.
[31] J. Berger, J. Hauber, R. Hauber, R. Geiger, and B.R. Cullen, Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66 (1988) 1-10.
[32] N. Hiramatsu, A. Kasai, Y. Meng, K. Hayakawa, J. Yao, and M. Kitamura, Alkaline phosphatase vs luciferase as secreted reporter molecules in vivo. Anal Biochem 339 (2005) 249-56.
[33] B.R. Cullen, and M.H. Malim, Secreted placental alkaline phosphatase as a eukaryotic reporter gene. Methods Enzymol 216 (1992) 362-8.
[34] A. Cimmino, G.A. Calin, M. Fabbri, M.V. Iorio, M. Ferracin, M. Shimizu, S.E. Wojcik, R.I. Aqeilan, S. Zupo, M. Dono, L. Rassenti, H. Alder, S. Volinia, C.G. Liu, T.J. Kipps, M. Negrini, and C.M. Croce, miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A 102 (2005) 13944-9.
[35] J.G. Doench, and P.A. Sharp, Specificity of microRNA target selection in translational repression. Genes Dev 18 (2004) 504-11.
[36] J. Brennecke, A. Stark, R.B. Russell, and S.M. Cohen, Principles of microRNA-target recognition. PLoS Biol 3 (2005) e85.
[37] G.A. Calin, C.D. Dumitru, M. Shimizu, R. Bichi, S. Zupo, E. Noch, H. Aldler, S. Rattan, M. Keating, K. Rai, L. Rassenti, T. Kipps, M. Negrini, F. Bullrich, and C.M. Croce, Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 99 (2002) 15524-9.
[38] A. Bottoni, D. Piccin, F. Tagliati, A. Luchin, M.C. Zatelli, and E.C. degli Uberti, miR-15a and miR-16-1 down-regulation in pituitary adenomas. J Cell Physiol 204 (2005) 280-5.
[39] D. Bonci, V. Coppola, M. Musumeci, A. Addario, R. Giuffrida, L. Memeo, L. D'Urso, A. Pagliuca, M. Biffoni, C. Labbaye, M. Bartucci, G. Muto, C. Peschle, and R. De Maria, The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nat Med 14 (2008) 1271-7.
[40] P.S. Linsley, J. Schelter, J. Burchard, M. Kibukawa, M.M. Martin, S.R. Bartz, J.M. Johnson, J.M. Cummins, C.K. Raymond, H. Dai, N. Chau, M. Cleary, A.L. Jackson, M. Carleton, and L. Lim, Transcripts targeted by the microRNA-16 family cooperatively regulate cell cycle progression. Mol Cell Biol 27 (2007) 2240-52.
[41] L. Xia, D. Zhang, R. Du, Y. Pan, L. Zhao, S. Sun, L. Hong, J. Liu, and D. Fan, miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human gastric cancer cells. Int J Cancer 123 (2008) 372-9.
[42] A.G. Letai, Diagnosing and exploiting cancer's addiction to blocks in apoptosis. Nat Rev Cancer 8 (2008) 121-32.
[43] T. Chittenden, C. Flemington, A.B. Houghton, R.G. Ebb, G.J. Gallo, B. Elangovan, G. Chinnadurai, and R.J. Lutz, A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO J 14 (1995) 5589-96.
[44] T. Kuwana, L. Bouchier-Hayes, J.E. Chipuk, C. Bonzon, B.A. Sullivan, D.R. Green, and D.D. Newmeyer, BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell 17 (2005) 525-35.
[45] B.D. Harfe, MicroRNAs in vertebrate development. Curr Opin Genet Dev 15 (2005) 410-5.
[46] H.W. Hwang, and J.T. Mendell, MicroRNAs in cell proliferation, cell death, and tumorigenesis. Br J Cancer 94 (2006) 776-80.
[47] W.P. Kloosterman, and R.H. Plasterk, The diverse functions of microRNAs in animal development and disease. Dev Cell 11 (2006) 441-50.
[48] R.J. Bold, S. Virudachalam, and D.J. McConkey, BCL2 expression correlates with metastatic potential in pancreatic cancer cell lines. Cancer 92 (2001) 1122-9.
[49] A. Frenzel, F. Grespi, W. Chmelewskij, and A. Villunger, Bcl2 family proteins in carcinogenesis and the treatment of cancer. Apoptosis 14 (2009) 584-96.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top