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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 阮雪芬(Hsueh-Fen Juan) | |
dc.contributor.author | Kuan-Hao Hsu | en |
dc.contributor.author | 徐寬豪 | zh_TW |
dc.date.accessioned | 2021-06-16T10:14:02Z | - |
dc.date.available | 2018-08-25 | |
dc.date.copyright | 2013-08-25 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-19 | |
dc.identifier.citation | Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-297 (2004).
2 Carleton, M., Cleary, M. A. & Linsley, P. S. MicroRNAs and cell cycle regulation. Cell cycle 6, 2127-2132 (2007). 3 Harfe, B. D. MicroRNAs in vertebrate development. Current opinion in genetics & development 15, 410-415, doi:10.1016/j.gde.2005.06.012 (2005). 4 Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858-862, doi:10.1126/science.1065062 (2001). 5 Boehm, M. & Slack, F. J. MicroRNA control of lifespan and metabolism. Cell cycle 5, 837-840 (2006). 6 Macfarlane, L. A. & Murphy, P. R. MicroRNA: Biogenesis, Function and Role in Cancer. Current genomics 11, 537-561, doi:10.2174/138920210793175895 (2010). 7 Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415-419, doi:10.1038/nature01957 (2003). 8 Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E. & Kutay, U. Nuclear export of microRNA precursors. Science 303, 95-98, doi:10.1126/science.1090599 (2004). 9 Hutvagner, G. et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834-838, doi:10.1126/science.1062961 (2001). 10 Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208 (2003). 11 Khvorova, A., Reynolds, A. & Jayasena, S. D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209-216 (2003). 12 He, L. & Hannon, G. J. MicroRNAs: small RNAs with a big role in gene regulation. Nature reviews. Genetics 5, 522-531, doi:10.1038/nrg1379 (2004). 13 Ferdin, J., Kunej, T. & Calin, G. A. Non-coding RNAs: identification of cancer-associated microRNAs by gene profiling. Technology in cancer research & treatment 9, 123-138 (2010). 14 Di Leva, G. & Croce, C. M. Roles of small RNAs in tumor formation. Trends in molecular medicine 16, 257-267, doi:10.1016/j.molmed.2010.04.001 (2010). 15 Li, C. et al. MiRNA-199a-3p: A Potential Circulating Diagnostic Biomarker for Early Gastric Cancer. Journal of surgical oncology, doi:10.1002/jso.23358 (2013). 16 Tseng, C. W., Lin, C. C., Chen, C. N., Huang, H. C. & Juan, H. F. Integrative network analysis reveals active microRNAs and their functions in gastric cancer. BMC systems biology 5, 99, doi:10.1186/1752-0509-5-99 (2011). 17 Lujambio, A. et al. A microRNA DNA methylation signature for human cancer metastasis. Proceedings of the National Academy of Sciences of the United States of America 105, 13556-13561, doi:10.1073/pnas.0803055105 (2008). 18 Chen, Y. et al. Altered expression of MiR-148a and MiR-152 in gastrointestinal cancers and its clinical significance. Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract 14, 1170-1179, doi:10.1007/s11605-010-1202-2 (2010). 19 Liffers, S. T. et al. MicroRNA-148a is down-regulated in human pancreatic ductal adenocarcinomas and regulates cell survival by targeting CDC25B. Laboratory investigation; a journal of technical methods and pathology 91, 1472-1479, doi:10.1038/labinvest.2011.99 (2011). 20 Zhang, H. et al. MiR-148a promotes apoptosis by targeting Bcl-2 in colorectal cancer. Cell death and differentiation 18, 1702-1710, doi:10.1038/cdd.2011.28 (2011). 21 Morin, R. et al. Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing. BioTechniques 45, 81-94, doi:10.2144/000112900 (2008). 22 Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nature reviews. Genetics 10, 57-63, doi:10.1038/nrg2484 (2009). 23 Clark, T. A., Sugnet, C. W. & Ares, M., Jr. Genomewide analysis of mRNA processing in yeast using splicing-specific microarrays. Science 296, 907-910, doi:10.1126/science.1069415 (2002). 24 David, L. et al. A high-resolution map of transcription in the yeast genome. Proceedings of the National Academy of Sciences of the United States of America 103, 5320-5325, doi:10.1073/pnas.0601091103 (2006). 25 Yamada, K. et al. Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 302, 842-846, doi:10.1126/science.1088305 (2003). 26 Bertone, P. et al. Global identification of human transcribed sequences with genome tiling arrays. Science 306, 2242-2246, doi:10.1126/science.1103388 (2004). 27 Cheng, J. et al. Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution. Science 308, 1149-1154, doi:10.1126/science.1108625 (2005). 28 Okoniewski, M. J. & Miller, C. J. Hybridization interactions between probesets in short oligo microarrays lead to spurious correlations. BMC bioinformatics 7, 276, doi:10.1186/1471-2105-7-276 (2006). 29 Royce, T. E., Rozowsky, J. S. & Gerstein, M. B. Toward a universal microarray: prediction of gene expression through nearest-neighbor probe sequence identification. Nucleic acids research 35, e99, doi:10.1093/nar/gkm549 (2007). 30 Gerhard, D. S. et al. The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome research 14, 2121-2127, doi:10.1101/gr.2596504 (2004). 31 Boguski, M. S., Tolstoshev, C. M. & Bassett, D. E., Jr. Gene discovery in dbEST. Science 265, 1993-1994 (1994). 32 Mardis, E. R. The impact of next-generation sequencing technology on genetics. Trends in genetics : TIG 24, 133-141, doi:10.1016/j.tig.2007.12.007 (2008). 33 Jemal, A. et al. Cancer statistics, 2004. CA: a cancer journal for clinicians 54, 8-29 (2004). 34 Yamazaki, H., Oshima, A., Murakami, R., Endoh, S. & Ubukata, T. A long-term follow-up study of patients with gastric cancer detected by mass screening. Cancer 63, 613-617 (1989). 35 Li, R. et al. De novo assembly of human genomes with massively parallel short read sequencing. Genome research 20, 265-272, doi:10.1101/gr.097261.109 (2010). 36 Wang, Z. et al. De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas). BMC genomics 11, 726, doi:10.1186/1471-2164-11-726 (2010). 37 Huang da, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature protocols 4, 44-57, doi:10.1038/nprot.2008.211 (2009). 38 Huang da, W., Sherman, B. T. & Lempicki, R. A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic acids research 37, 1-13, doi:10.1093/nar/gkn923 (2009). 39 Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature methods 5, 621-628, doi:10.1038/nmeth.1226 (2008). 40 Benjamini, Y., Drai, D., Elmer, G., Kafkafi, N. & Golani, I. Controlling the false discovery rate in behavior genetics research. Behavioural brain research 125, 279-284 (2001). 41 Shiloh, Y. ATM and ATR: networking cellular responses to DNA damage. Current opinion in genetics & development 11, 71-77 (2001). 42 Malumbres, M. & Barbacid, M. Mammalian cyclin-dependent kinases. Trends in biochemical sciences 30, 630-641, doi:10.1016/j.tibs.2005.09.005 (2005). 43 Chang, X. et al. Ligand-independent regulation of transforming growth factor beta1 expression and cell cycle progression by the aryl hydrocarbon receptor. Molecular and cellular biology 27, 6127-6139, doi:10.1128/MCB.00323-07 (2007). 44 Elizondo, G. et al. Altered cell cycle control at the G(2)/M phases in aryl hydrocarbon receptor-null embryo fibroblast. Molecular pharmacology 57, 1056-1063 (2000). 45 Brown, J. R. et al. Fos family members induce cell cycle entry by activating cyclin D1. Molecular and cellular biology 18, 5609-5619 (1998). 46 Lin, D. et al. Constitutive expression of B-myb can bypass p53-induced Waf1/Cip1-mediated G1 arrest. Proceedings of the National Academy of Sciences of the United States of America 91, 10079-10083 (1994). 47 Yamaguchi, F. et al. Rare sugar D-allose induces specific up-regulation of TXNIP and subsequent G1 cell cycle arrest in hepatocellular carcinoma cells by stabilization of p27kip1. International journal of oncology 32, 377-385 (2008). 48 Wyllie, A. H., Kerr, J. F. & Currie, A. R. Cell death: the significance of apoptosis. International review of cytology 68, 251-306 (1980). 49 Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555-556 (1980). 50 Nagata, S. Apoptotic DNA fragmentation. Experimental cell research 256, 12-18, doi:10.1006/excr.2000.4834 (2000). 51 Suzuki, T., Fujikura, K., Higashiyama, T. & Takata, K. DNA staining for fluorescence and laser confocal microscopy. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 45, 49-53 (1997). 52 Molinari, M. Cell cycle checkpoints and their inactivation in human cancer. Cell proliferation 33, 261-274 (2000). 53 Hartwell, L. H. & Kastan, M. B. Cell cycle control and cancer. Science 266, 1821-1828 (1994). 54 Krek, W. & Nigg, E. A. Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates. The EMBO journal 10, 3331-3341 (1991). 55 Malumbres, M. & Barbacid, M. Cell cycle, CDKs and cancer: a changing paradigm. Nature reviews. Cancer 9, 153-166, doi:10.1038/nrc2602 (2009). 56 Coller, H. A. What's taking so long? S-phase entry from quiescence versus proliferation. Nature reviews. Molecular cell biology 8, 667-670, doi:10.1038/nrm2223 (2007). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60236 | - |
dc.description.abstract | 微型核醣核酸為一種長約20~22鹼基小片段內生型核醣核酸,本身並不會轉錄出蛋白質,但會藉由結合標靶訊息核糖核酸抑制蛋白質的轉抑或是造成訊息核糖核酸的降解,進而調控基因表現。本實驗利用次世代定序RNA-seq研究微型核醣核酸148a對於胃癌細胞AGS的轉錄組造成的影響,進而探討微型核糖核酸148a可能尚未被發現的功能。從RNA-seq的資料中獲得了16,254個表現差異基因,利用基因表達量(RPKM)、基因表現量差異、錯誤發現率(FDR)來篩選顯著表現差異之基因,其中,有63個表現上升和48個表現下降的基因被挑選出來。藉由生物資訊工具,以基因本體(gene ontology)、基因功能性分析RNA-seq所挑選出的基因,得到前兩名結果分別為細胞死亡及細胞週期。在細胞集落形成測定法以及生長曲線監控實驗中,微型核糖核酸148a會抑制AGS細胞生長。利用4',6-二脒基-2-苯基吲哚(DAPI)染色在螢光顯微鏡的觀察下,並沒有發現DNA聚合、細胞凋亡情況發生;利用流式細胞儀,我們發現轉染微型核醣核酸148a的AGS細胞,在細胞週期G2/M族群分布增加。除此之外,磷酸化CDC2 (Thr-161)的蛋白質表現量下降,CDK4/6、Cyclin D1及磷酸化Rb的蛋白質表現量上升之結果,造成G1細胞周期活化進而使G2/M細胞週期累積有關。此實驗結果顯示,藉由RNA-seq揭露微型核糖核酸148a扮演調控細胞週期G1的新角色,可能提供了對於治療胃癌具有潛力的資訊。 | zh_TW |
dc.description.abstract | MicroRNAs play an important role in various biological processes by post-transcriptionally regulating gene expression. To investigate the role of miR-148a in gastric cancer, the next generation RNA sequencing (RNA-seq) was applied to reveal the miR-148a-regulated gene expression profiles in gastric cancer cells. To uncover the role of miR-148a in gastric cancer, miR-148a was overexpressed in gastric cancer cell line AGS and followed by RNA-seq analysis. Using RNA-seq analysis, gene expression levels were estimated to identify differentially expressed genes in miR-148a-overexpressed cells. The biological processes and functional networks associated with the regulated genes were further analyzed by Ingenuity Pathway analysis (IPA). To validate the finding from RNA-seq, the expression level of the interested genes was measured by real-time reverse transcription PCR (qRT-PCR), the cell growth was monitored in real-time using the xCELLigence system, and the cell cycle analysis was examined by flow cytometry.
We identified a total of 16,254 genes by comparing RNA-seq profiles of negative control (NTC) and miR-148a-overexpressed (Pre) AGS cells. Among them, 63 up-regulated and 48 down-regulated genes were differentially expressed in miR-148a-overexpressed cells. The IPA results showed that these miR-148a-regulated genes were significantly involved in the biological pathways including cell death and cell cycle. We demonstrated overexpression of miR-148a reduced the cell growth and alter the cell cycle G2/M phase population increased. This study illustrates the new role of miR-148a in controlling cell cycle by RNA-seq and provides information in gastric cancer therapy. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:14:02Z (GMT). No. of bitstreams: 1 ntu-102-R00b43024-1.pdf: 3097230 bytes, checksum: e248c7ec184df7431795afffc6bda45d (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | Contents
中文摘要 I Abstract II 致謝 IV Figure contents VIII Table contents IX Chapter 1 Introduction 1 1.1 miRNA and miR-148a 1 1.2 RNA-sequencing 3 1.3 Gastric cancer 6 1.4 Experimental design 6 Chapter 2 Material and Methods 8 2.1 Cell culture 8 2.2 Transfection 8 2.3 RNA extraction 9 2.4 RNA integrity examination and RNA-seq library preparation 10 2.5 Bioinformatics analysis – assembly and functional annotation 11 2.6 Difference in gene expression profile between Pre and NTC 13 2.7 Total RNA reverse transcription 14 2.8 miRNA reverse transcription 14 2.9 SYBR real-time PCR 15 2.10 TaqMan real-time PCR 16 2.11 DAPI staining 16 2.12 Apoptosis detection 17 2.13 Cell cycle analysis by flow cytometry 17 2.14 Protein extraction and Western blotting 18 2.15 Colony formation 19 Chapter 3 Results 20 3.1 The RNA-sequencing of gastric cancer cells with miR148a overexpression 20 3.2 Analysis of differentially expressed genes in miR-148a over-expressing cells 20 3.3 Functional analysis of miR-148a regulated genes 21 3.4 Validation of the gene expression in RNA-seq data by qPCR analysis 22 3.5 miR-148a overexpression does not induce the cell apoptosis in AGS cells 24 3.6 miR-148a overexpression alters the cell cycle distribution in AGS cells 24 3.7 miR-148a overexpression has no effect on the expression level of G2/M regulated genes in gastric cancer cells 25 3.8 miR-148a overexpression alters the protein expression level of G1 regulated genes 26 3.9 miR-148a inhibits the growth of gastric cancer cells 27 Chapter 4 Discussion 29 Reference 32 Figures 41 Table 52 Appendix 61 | |
dc.language.iso | en | |
dc.title | 以高通量定序方式解析微型核醣miR-148a於胃癌AGS細胞所扮演的角色 | zh_TW |
dc.title | Elucidating the Role of miR-148a in Gastric Cancer AGS Cells Using RNA-seq | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃宣誠(Hsuan-Cheng Huang),溫進德(Jin-Der Wen),李岳倫(Yueh-Luen Lee),黃翠琴(Tsui-Chin Huang) | |
dc.subject.keyword | 微型核醣核酸,胃癌,RNA-seq,IPA,細胞週期, | zh_TW |
dc.subject.keyword | miRNA,gastric cancer,RNA-seq,IPA,cell cycle, | en |
dc.relation.page | 62 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-19 | |
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 |
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