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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 蔡懷楨(Huai-Jen Tsai) | |
dc.contributor.author | Ming-Hsuan Lee | en |
dc.contributor.author | 李明軒 | zh_TW |
dc.date.accessioned | 2021-06-08T00:02:08Z | - |
dc.date.copyright | 2013-08-27 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-15 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17231 | - |
dc.description.abstract | 微型核醣核酸 microRNAs (miRNAs) 為小片段非轉譯之單股核醣核酸,其藉由保守性種子序列 (conserved seed sequence) 與標的基因mRNA之3’端非轉譯區 (3’-untranslated region,3’UTR),於轉錄後層次 (post-transcriptional level) 調控目標基因的表現。MicroRNA-1 (miR-1) 與microRNA-206 (miR-206) 同樣為肌肉專一型微型核醣核酸 (muscle-specific miRNA),因為具有完全相同的保守性種子序列,推測兩者能調控相同的目標基因,但仍不清楚。因此,我們利用 Labeled microRNA pull-down (LAMP) assay system 篩選48 hpf胚胎mRNA,並配合 microarray assay 分析。結果發現在斑馬魚胚胎中miR-1能與seryl-tRNA synthetase (sars) mRNA的3’UTR結合。接著利用Dual-luciferase reporter assay於斑馬魚胚胎內證實,miR-1能夠專一地透過sars-3’UTR抑制報導基因的表現,而miR-206卻不能。並且由西方浸漬法證明只有當 miR-1 被抑制時斑馬魚內生性 SARS蛋白質的表現量會增加,但抑制miR-206時胚胎內SARS蛋白質的表現量不變。另一方面,當胚胎中降低miR-1表現會造成其血管內皮生長因子 (VegfAa) 表現量降低且體節間血管 (ISV) 生長遲緩,無法正常向上延伸至背部;而當降低胚胎內miR-206時,卻會造成VegfAa蛋白質表現量上升且ISV內皮細胞異常增生、分歧。進一步觀察抑制miR-1或過量表現 sars mRNA的胚胎其細胞核內外SARS蛋白質表現量皆增加,同時會使斑馬魚vegfaa啟動子 (-1434~+640 nt) 驅動活性降低。此外,透過人類胚腎細胞株HEK293T進行Dual-luciferase reporter assay,發現於不同的物種間,SARS抑制vegfa啟動子活性的能力具有保守性;且人類SARS T429A可藉由人類vegfa啟動子-1180∼-764 nt序列負向調控人類vegfa啟動子的驅動活性。綜合以上實驗結果,我們認為miR-1與miR-206對於血管發育擁有截然不同的影響:miR-1透過默化sars而促進vegfaa啟動子活性與血管新生,然而miR-206卻是抑制vegfaa而阻礙血管新生。兩者於胚胎發育過程中共同維持血管正常生長之發育。 | zh_TW |
dc.description.abstract | MicroRNAs (miRNAs) are short, endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level by targeting the 3’-untranslated region (3’UTR) of mRNAs through a conserved seed sequence. MicroRNA-1 (miR-1) and microRNA-206 (miR-206) are muscle–specific miRNAs. Since miR-1 and miR-206 have identical conserved seed sequence, they are commonly speculated to have the same target genes. However, it is unclear whether they do silence the same target gene during embryogenesis. Hence, we employed Labeled microRNA pull-down (LAMP) assay system and microarray assay to screen putative mRNA targets for miR-1 from whole-cell extracts of 48-hpf zebrafish embryos. We obtained a mRNA encoded Seryl-tRNA Synthetase (SARS) whose 3’UTR was specifically repressed by miR-1, but not miR-206, in muscle cells of zebrafish. Using Western blot analysis, we found that knockdown of miR-1 increased the protein level of endogenous SARS in zebrafish embryos, while knockdown of miR-206 did not change the SARS protein level. Besides, knockdown of miR-1 in zebrsfish embryos also caused the decrease of vascular endothelial growth factor Aa (VegfAa) protein, which further led to lose organization and delay development of intersegmental vessels (ISV). However, we observed that increase of VegfAa protein and ectopically branching ISV accompanied with a proliferation of endothelial cells in the miR-206 morphants. Furthermore, we observed that the nuclear content of SARS protein was increased in the embryos injected both with miR-1-MO and sars mRNA, resulting in down-regulation of the promoter activity of zebrafish vegfaa. Moreover, the negative control of SARS in vegfa is functionally conserved between zebrafish and humans. Additionally, human SARS T429A repressed luciferase activity through the sequence segment from -1180 to -764 nucleotides of human vegfa promoter. Taken together, we conclude that miR-1 and miR-206 play different roles on the zebrafish vascular development, although both of them contribute to normal angiogenesis in zebrafish embryos. That is miR-1 enhances vegfaa promoter activity and promotes angiogenesis through silencing sars; whereas miR-206 inhibits angiogenesis through repressing vegfaa. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T00:02:08Z (GMT). No. of bitstreams: 1 ntu-102-R00b43025-1.pdf: 3106655 bytes, checksum: 86559b316e7183a15ee14d5863398207 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 中文摘要---------------------------------------------------------------------------1
英文摘要---------------------------------------------------------------------------2 文獻回顧---------------------------------------------------------------------------4 前言--------------------------------------------------------------------------------19 材料與方法-----------------------------------------------------------------------22 結果--------------------------------------------------------------------------------34 討論--------------------------------------------------------------------------------44 參考文獻--------------------------------------------------------------------------50 圖表--------------------------------------------------------------------------------69 補充圖表--------------------------------------------------------------------------81 附錄--------------------------------------------------------------------------------83 | |
dc.language.iso | zh-TW | |
dc.title | microRNA-1與microRNA-206調控斑馬魚血管新生之分子機制 | zh_TW |
dc.title | Molecular mechanisms of microRNA-1 and microRNA-206 on regulating zebrafish angiogenesis | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王健家,劉薏雯,詹世鵬 | |
dc.subject.keyword | 斑馬魚,微型核醣核酸,microRNA-1,microRNA-206,血管新生,tRNA合成酶, | zh_TW |
dc.subject.keyword | microRNAs,microRNA-1,microRNA-206,angiogenesis,zebrafish,seryl-tRNA synthetase, | en |
dc.relation.page | 88 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2013-08-15 | |
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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