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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 阮雪芬(Hsueh-Fen Juan) | |
| dc.contributor.author | Shih-Han Lin | en |
| dc.contributor.author | 林詩涵 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:27:32Z | - |
| dc.date.available | 2028-12-31 | |
| dc.date.copyright | 2018-08-16 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-13 | |
| dc.identifier.citation | 1. Huang, M. and Weiss, W.A. (2013) Neuroblastoma and MYCN. Cold Spring Harb. Perspect. Med., 3, a014415.
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(2013) miR-203 inhibits cell proliferation and migration of lung cancer cells by targeting PKCα. PloS One, 8, e73985. 64. Yang, T.-W. (2017) Long non-coding RNA SNHG1 in neuroblastoma. Master Thesis, National Taiwan University. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70408 | - |
| dc.description.abstract | 神經母細胞瘤(neuroblastoma)是一種好發於兒童的癌症，為一種常見的胚胎腫瘤，源自於未分化的神經嵴細胞。大約25％的神經母細胞瘤患者發生MYCN轉錄基因異常放大，導致高風險疾病和不良預後。近年來，許多研究指出長鏈非編碼核醣核酸(long noncoding RNA)在腫瘤的形成過程中會扮演許多重要的角色。長鏈非編碼核醣核酸為長200餘鹼基對似訊息核醣核酸，但不會轉譯出蛋白質，其功能之一為引導蛋白質(例如：轉錄因子)到它的標的蛋白質。然而，由MYCN轉錄基因異常放大所調控的長鏈非編碼核醣核酸，其與神經母細胞瘤的高風險族群及不良預後之間的關係仍不明確。在我們實驗室之前的研究中，從MYCN轉錄基因擴增(n=92)及MYCN轉錄基因非擴增(n=401)的腫瘤中，結合微陣列(microarray)與核醣核酸測序(RNA-sequencing) 的長鏈非編碼核醣核酸基因表現調控資料庫，進行網絡整合分析，我們發現small nucleolar RNA host gene 1 (SNHG1)的表現量差異和MYCN轉錄基因異常放大表現量呈現高度正相關性，並利用即時聚合酶鍊式反應驗證之。在本研究中，為了研究SNHG1的調控機制，我們運用小分子干擾核糖核酸 (siRNA) 以及基因編輯技術CRISPR/Cas9在神經母細胞瘤細胞中敲落或刪除SNHG1的基因位點，減弱或剔除SNHG1會影響細胞增生。我們發現MYCN會調控SNHG1的啟動子，且抑制MYCN的表現量後，SNHG1的表現量也隨之下降；另外，有先前研究指出，SNHG1會調控MYC，其和MYCN有補償作用，也就是說，敲落SNHG1的表現量後，其會抑制MYC的表現量，但卻促進MYCN的表現量，這也是敲落SNHG1的表現量後，造成細胞大量增生的原因之一。我們進一步整合SNHG1干預的蛋白體和轉錄體學去了解其下游調控因子及其參與的生物途徑，研究發現SNHG1影響的功能集中在細胞增殖，細胞粘附和細胞遷移。我們也以細胞遷移分析實驗、即時聚合酶鍊式反應和西方墨點法證實剔除SNHG1會增加與遷移有關的蛋白質L1CAM, EGFR 和PRKCA表現，而促進細胞遷移能力。這些結果表示SNHG1參與調節MYCN和MYC的反饋迴路中，並促進有助於神經母細胞瘤腫瘤發生的細胞遷移。 | zh_TW |
| dc.description.abstract | Neuroblastoma is a common embryonal tumor originating from undifferentiated neural crest cells. Approximately 25% patients with neuroblastoma have MYCN oncogene amplification resulting in high-risk disease and poor prognosis. Recently, long noncoding RNAs (lncRNAs) are reported to have various roles in tumorigenesis. Long noncoding RNAs constitute major proportion of the cellular transcripts with no coding capacity. One of their functions is to guide transcription factors to the target genes and facilitate gene expression. However, lncRNAs that are altered by MYCN amplification, associated with high-risk, and disease prognosis remain unclear. We performed a network-based integrative analysis from both microarray and RNA-seq lncRNA expression profiles between MYCN amplified (n = 92) and MYCN non-amplified (n = 401) tumor subtypes. We identified the differentially expressed lncRNA small nucleolar RNA host gene 1 (SNHG1) to be significantly correlated with MYCN-amplification, and was further validated by RT-qPCR. Here, to study the regulatory function by SNHG1, we performed small interfering RNAs (siRNA) targeting SNHG1 and CRISPR/Cas9 editing to delete the genomic locus of SNHG1 in neuroblastoma cells, while knockdown or knockout of SNHG1 increased cell proliferation. We found MYCN binds to the promoter of SNHG1 and silencing MYCN downregulated SNHG1 expression. Additionally, previous studies have reported that SNHG1 regulated MYC which compensates MYCN. Thence, knockdown of SNHG1 would decrease MYC expression but enhance MYCN expression, which was the reason why knockdown of SNHG1 increased cell proliferation. We further integrated SNHG1-mediated proteomics and transcriptomics to reveal the downstream regulators and its biological processes. Our investigation found the functional enrichment on cell proliferation, cell adhesion and cell migration. Moreover, we confirmed that knockout of SNHG1 increased the mRNA and protein expressions of three migration markers, L1CAM, EGFR and PRKCA, and promote cell migration using cell migration assay, RT-qPCR and western blot. These results suggest that SNHG1 participates in a feedback loop regulating MYC and MYCN and promoted cell migration that contributes to neuroblastoma tumorigenesis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:27:32Z (GMT). No. of bitstreams: 1 ntu-107-R05b43007-1.pdf: 4630025 bytes, checksum: 55312e263ea012395ff56e70530da0ba (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員審定書 i
致謝 ii 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 avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) 3 1.3 Gene expression analysis 4 1.4 Long noncoding RNA 5 1.5 Small nucleolar RNA host gene 1 (SNHG1) 7 1.6 CRISPR/ Cas system 8 1.7 Motivation 9 Chapter 2. Materials and Methods 10 2.1 Experimental design 10 2.2 Cell culture 10 2.3 Transient siRNA knockdown of SNHG1 11 2.4 Construction of plasmids 11 2.5 Transfection of plasmid DNAs and selection 12 2.6 RNA extraction and reverse transcription 12 2.7 Real-time quantitative polymerase chain reaction (RT-qPCR) 14 2.8 Genomic DNA isolation 15 2.9 Polymerase chain reaction (PCR) 16 2.10 Cell viability assay 17 2.11 Colony formation assay 17 2.12 Protein precipitation 18 2.13 Reduction, alkylation and digestion of proteins 19 2.14 Dimethyl labeling of peptides 20 2.15 Desalting with SDB-XC Stage Tips 21 2.16 SCX fractionation 21 2.17 NanoLC-MS/MS analysis 22 2.18 Proteome data analysis 23 2.19 RNA integrity examination and RNA-seq library preparation 24 2.20 Western blot 25 2.21 Cell migration assays 26 Chapter 3. Results 27 3.1 Targeted deletion of SNHG1 by CRISPR/ Cas9 system 27 3.2 Knockdown of SNHG1 increased cell proliferation 29 3.3 SNHG1 regulates MYC which compensates MYCN in neuroblastoma 30 3.4 The RNA-sequencing of neuroblastoma cells with SNHG1 deletion 31 3.5 Biological process analysis with SNHG1-deregulated by transcriptomics in neuroblastoma cells 33 3.6 Quantitative proteomic analysis of SNHG1-deletion in neuroblastoma cells 33 3.7 Functional enrichments and biological processes of SNHG1-mediated proteomics 35 3.8 Integrative analysis of SNHG1-regulatory proteome and RNA-seq in neuroblastoma cells 36 3.9 Validation of SNHG1 functional networks on cell migration in neuroblastoma cells 38 Chapter 4. Discussion 40 Chapter 5. Conclusion 45 Reference 47 Tables 57 Figures 86 | |
| dc.language.iso | en | |
| dc.subject | 長鏈非編碼核醣核酸 | zh_TW |
| dc.subject | MYCN | zh_TW |
| dc.subject | 神經母細胞瘤 | zh_TW |
| dc.subject | 細胞遷移 | zh_TW |
| dc.subject | 核醣核酸測序 | zh_TW |
| dc.subject | SNHG1 | zh_TW |
| dc.subject | 液相層析串聯式質譜儀 | zh_TW |
| dc.subject | cell migration | en |
| dc.subject | MYCN | en |
| dc.subject | Long non-coding RNA | en |
| dc.subject | SNHG1 | en |
| dc.subject | LC-MS/MS | en |
| dc.subject | RNA-seq | en |
| dc.subject | Neuroblastoma | en |
| dc.title | 探討長鏈非編碼核糖核酸SNHG1在人類神經母細胞瘤的癌化機制 | zh_TW |
| dc.title | Long Non-coding RNA SNHG1 in the Regulation of Neuroblastoma Tumorigenesis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃宣誠(Hsuan-Cheng Huang),黃敏銓(Min-Chuan Huang),許文明(Wen-Ming Hsu),許家郎(Chia-Lang Hsu) | |
| dc.subject.keyword | 神經母細胞瘤,MYCN,長鏈非編碼核醣核酸,SNHG1,液相層析串聯式質譜儀,核醣核酸測序,細胞遷移, | zh_TW |
| dc.subject.keyword | Neuroblastoma,MYCN,Long non-coding RNA,SNHG1,LC-MS/MS,RNA-seq,cell migration, | en |
| dc.relation.page | 108 | |
| dc.identifier.doi | 10.6342/NTU201803210 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-14 | |
| 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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