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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 | Ya-Chih Fan | en |
dc.contributor.author | 范雅智 | zh_TW |
dc.date.accessioned | 2021-06-16T05:09:18Z | - |
dc.date.available | 2025-07-29 | |
dc.date.copyright | 2020-08-03 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-07-29 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55839 | - |
dc.description.abstract | Small nucleolar RNA host gene 1 (SNHG1) 是一種新穎的致癌性長鏈非編碼核醣核酸 (long noncoding RNA) ,在不同腫瘤類型中異常表達。在我們實驗室之前的研究中發現,高表達的SNHG1與好發在兒童顱外的神經母細胞瘤 (Neuroblastoma) 的預後不良及MYCN狀態有關。然而,SNHG1在神經母細胞瘤中的分子機制還尚未明確。為了研究SNHG1在神經母細胞瘤中作用,我們利用基因編輯技術CRISPR/Cas9干擾內源性SNHG1表現在MYCN基因異常放大的SK-N-BE(2)C神經母細胞瘤細胞中。確實敲弱SNHG1的表現會抑制細胞增殖與集落形成的能力。在敲弱SNHG1的細胞中,透過RNA-sequencing、RNA結合蛋白體學和功能分析發現,耗盡SNHG1會抑制細胞生長和遷移,並誘導細胞凋亡、細胞週期G1期的停滯、與活性氧 (ROS) 產生。有趣的是,在敲弱SNHG1的細胞中,會降低MYCN基因異常放大的神經母細胞瘤內的核心轉錄調控電路 (CRC) 成員的水平表現,包括PHOX2B,HAND2,GATA3,ISL1,TBX1和MYCN。透過抗體-H3K27ac染色質免疫沉澱測序和染色質開放性測序分析,發現這些CRC成員染色質狀態的改變,並且證明SNHG1直接與HDAC1/2相互作用,進而影響染色質的乙醯化水平。這些發現表明,SNHG1透過調節染色質狀態來維持在神經母細胞瘤中的身分扮演著至關重要的角色。總結來說,此篇研究揭示了長鏈非編碼核醣核酸SNHG1在神經母細胞瘤中的功能。 | zh_TW |
dc.description.abstract | The small nucleolar RNA host gene 1 (SNHG1) is a novel oncogenic long non-coding RNA (lncRNA) aberrantly expressed in different tumor types. In our previous work, highly expressed SNHG1 is associated with the poor-prognosis and MYCN status in neuroblastoma (NB), the most common extra-cranial solid tumor in children. However, the molecular mechanisms of SNHG1 in neuroblastoma is still unclear. To investigate the role of SNHG1 in neuroblastoma, we disrupted the endogenous SNHG1 of the MYCN-amplified neuroblastoma cell line SK-N-BE(2)C using the CRISPR/Cas9 system. The knock-down of SNHG1 indeed suppresses cell proliferation and colony formation. The RNA-sequencing, RNA-binding proteomics and functional assays for SNHG1-knockdown cells demonstrate the depletion of SNHG1 suppresses cell growth and migration, and induces apoptosis, G1-phase cell cycle arrest, and reactive oxygen species (ROS) production. Interestingly, the decreased expression levels of the core regulatory circuitry (CRC) members of MYCN-amplified NB, including PHOX2B, HAND2, GATA3, ISL1, TBX1, and MYCN, are observed in SNHG1-knockdown cells. The anti-H3K27ac chromatin-immunoprecipitation coupled to sequencing (ChIP-seq) and transposase-accessible chromatin using sequencing (ATAC-seq) analyses explore that the chromatin status of these CRC members are changed, and SNHG1 directly interacted with HDAC1/2 to affect chromatin acetylation level. These findings demonstrate the SNHG1 plays a crucial role in the maintenance of NB identity via regulation of chromatin status. In summary, this study reveals the lncRNA SNHG1 function in neuroblastoma. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T05:09:18Z (GMT). No. of bitstreams: 1 U0001-2807202018233400.pdf: 3585004 bytes, checksum: 6da5a7c3841a6e1a4f2a0db780eeef3c (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 致謝 i 中文摘要 iii Abstract v Contents vii List of Tables xi List of Figures xii Chapter 1. Introduction 1 1.1 Neuroblastoma 1 1.1.1 Tumor stage 2 1.2 v-myc avain myelocytomatosis viral oncogene neuroblastoma derived homolog 3 1.3 Long noncoding RNA 4 1.4 Small nucleolar host gene 1 (SNHG1) 5 1.5 CRISPR/Cas9 system 6 1.6 Multi-omics 7 1.7 Motivation 10 Chapter 2. Materials and Methods 11 2.1 Experimental design 11 2.2 Cell culture 11 2.3 Transient siRNA knockdown in SK-N-BE(2)C 11 2.4 CRISPR/Cas9 12 2.5 RNA extraction and reverse transcription 12 2.6 Real-time quantitative polymerase chain reaction (RT-qPCR) 14 2.7 Cell viability assay 14 2.8 Colony formation assay 15 2.9 Cell apoptosis assessments 15 2.10 Cell cycle analysis 15 2.11 Cell migration assay 16 2.12 Reaction oxygen species (ROS) assay 16 2.13 Western blot 17 2.14 Construction of SNHG1 overexpression plasmid 18 2.14.1 PCR amplification 18 2.14.2 Restriction enzyme digestion 19 2.14.3 Gel extraction 20 2.14.4 Ligation 20 2.14.5 Transformation 21 2.14.6 Colony PCR 21 2.14.7 Plasmid DNA purification 22 2.15 RNA-protein pull-down assay 23 2.15.1 In vitro transcription 23 2.15.2 Lithium chloride precipitation 24 2.15.3 RNA 3’ end desthiobiotinylation 24 2.15.4 Protein precipitation 25 2.15.5 RNA-Protein Pull-Down assay 26 2.16 RNA immunoprecipitation (RIP) 27 2.16.1 Lysate preparation 27 2.16.2 RNA-protein complexes immunoprecipitation 27 2.16.3 RNA purification 28 2.16.4 Reverse transcription-polymerase chain reaction (RT-PCR) 29 2.17 Sequencing analysis 30 2.17.1 RNA integrity examination and RNA-seq library preparation 30 2.17.2 Assay for transposase-accessible chromatin (ATAC)-sequencing 30 2.17.3 Chromatin immunoprecipitation (ChIP)-sequencing 32 Chapter 3. Results 33 3.1. Silence of SNHG1 suppressed neuroblastoma proliferation 33 3.2. Targeted deletion of SNHG1 by CRISPR/Cas9 system 33 3.3. SNHG1-depletion suppressed cell growth 34 3.4. The RNA-sequencing of SNHG1 depletion in neuroblastoma cells 34 3.5. SNHG1-depletion induced cell apoptosis in neuroblastoma cells 35 3.6. SNHG1-depletion induced G1-phase cell cycle arrest in neuroblastoma cells 35 3.7. SNHG1-depletion suppressed cell migration in neuroblastoma 36 3.8. SNHG1-depletion induced reactive oxygen species (ROS) in neuroblastoma cells 36 3.9. SNHG1-depletion decreased the transcription factors expression of core regulatory circuit (CRC) in neuroblastoma cells 37 3.10. SNHG1-depletion changed the chromatin state in neuroblastoma cells 38 3.11. SNHG1 maintained chromatin state via HDAC1/2 38 Chapter 4. Discussion 39 Chapter 5. Conclusion 42 References 44 Tables 53 Figures 76 | |
dc.language.iso | en | |
dc.title | 整合基因體編輯和多體學揭露長鏈非編碼核醣核酸SNHG1在神經母细胞瘤中的功能 | zh_TW |
dc.title | Integrative genome editing and multi-omics reveal lncRNA SNHG1 function in neuroblastoma | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃宣誠(Hsuan-Cheng Huang),許文明(Wen-Ming Hsu),蔡懷寬(Huai-Kuang Tsai),許家郎(Chia-Lang Hsu) | |
dc.subject.keyword | 長鏈非編碼核醣核酸,SNHG1,神經母細胞瘤,MYCN,RNA-sequencing,蛋白體學,細胞生長,細胞遷移,細胞凋亡,細胞週期,活性氧停滯,核心轉錄調節電路,染色質免疫沉澱測序,染色質開放性測序,HDAC1,HDAC2, | zh_TW |
dc.subject.keyword | Long non-coding RNA,SNHG1,Neuroblastoma,MYCN,RNA-sequencing,proteomic,cell growth,migration,apoptosis,cell cycle arrest,reactive oxygen species,core regulatory circuitry,chromatin-immunoprecipitation coupled to sequencing,transposase-accessible chromatin using sequencing,HDAC1,HDAC2, | en |
dc.relation.page | 96 | |
dc.identifier.doi | 10.6342/NTU202001993 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-07-30 | |
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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