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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊泮池(Pan-Chyr Yang),張逸良(Yih-Leong Chang) | |
| dc.contributor.author | An-Yi Hsiao | en |
| dc.contributor.author | 蕭安益 | zh_TW |
| dc.date.accessioned | 2021-06-08T00:55:04Z | - |
| dc.date.copyright | 2015-03-12 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-02-24 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18212 | - |
| dc.description.abstract | 胰臟癌是高度惡性的疾病，大多數患者出現明顯症狀時已屬晚期，往往伴隨著淋巴血管侵襲 (lymphovascular invasion) 以及轉移 (metastasis)，其存活率並不樂觀。本實驗室從惡性胰管腺癌 (ductal adenocarcinoma) 病患中建立了兩株胰臟癌細胞株，分別是 PK1 (derived from primary tumor)及PK2 (derived from metastatic site)。透過核醣核酸定序 (RNA Sequencing) 做廣泛的RNA level分析，而此研究探討的是其中“長鏈非編碼RNA (long non-coding RNA, LncRNA)”的部份。我們發現 NEAT1 在定序結果中不僅相對上讀值較高，且在兩者之間的表現量有顯著差異；利用 RT-qPCR 證實 NEAT1 在 PK2 的表現量確實高於 PK1。因此，為了了解 NEAT1 在胰臟癌轉移中的功能，初步使用 siRNA 的方式 knockdown NEAT1 在 PK2細胞中的表現量，並逐步檢測是否影響 EMT marker 的mRNA及蛋白質的表現量。在 NEAT1 表現量受到抑制的 PK2 細胞中，發現 Slug, Snail, Vimentin 的mRNA及蛋白表現量明顯減少，而 E-cadherin 表現量增加。進一步進行功能檢測：與控制組相比，當 siRNA降低 NEAT1 表現時，PK2 細胞在 wound healing assay 以及single cell tracking中的移動能力有顯著的下降；從invasion assay也可觀察到NEAT1 表現降低時會使得細胞侵襲能力下降。而且不只在PK2 knockdown NEAT1會影響其細胞移動及侵襲，我們也在肺癌細胞株CL1-5驗證，不論是phenotype change或是分子層級的表現都得到同樣的結果。除此之外，抑制NEAT1在胰臟癌細胞株AsPC-1/PL45及大腸癌細胞株HCT-15也會看到相同的phenotype change。綜合本研究結果，NEAT1 確實在癌移動與侵襲過程中扮演某些角色，但其中分子作用機制仍有待更進一步之研究證實。 | zh_TW |
| dc.description.abstract | Pancreatic cancer is a lethal disease needing vigorous bio-medical research. We established pancreatic cancer cell lines, PK1 and PK2, from a patient with poorly-differentiated ductal adenocarcinoma (PDAC). In order to dig out novel genetic alterations and potential treatment targets, genome-wide studies were carried out. The RNA-seq study showed that, NEAT1 long non-coding RNA was upregulated in PK2 cells as compared to PK1 cells. The RNA-seq results were verified by quantitative real-time RT-PCR (RT-qPCR). We employed small interfering/hairpin RNA knockdown of NEAT1 in PK2 cells. The results showed that the migration and invasion abilities of PK2 cells were reduced with NEAT1 knockdown. These findings were supported by the RT-qPCR and immunoblotting results in that the expression of epithelial-mesenchymal transition (EMT)-related markers, such as SNAIL, SLUG, and Vimentin, were suppressed while the expression of E-cadherin was increased in both transcript and protein levels with NEAT1 knocked down. Furthermore, NEAT1 knockdown also resulted in similar EMT marker alterations as well as reduced cell migration and invasion abilities in CL1-5 lung cancer cells. In addition, the same phenotypicale changes were observed in AsPC-1, PL45 and HCT-15 cells. These results suggest that NEAT1 may play a certain role in cancer migration and invasion. Detail mechanisms and involving signaling pathways deserved further studies. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T00:55:04Z (GMT). No. of bitstreams: 1 ntu-104-R01444005-1.pdf: 3914078 bytes, checksum: a79ba1d94a42d962fdc8f20f7d2f4f8f (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 目錄 口試委員審定書……………………………………………………………………# 中文摘要……………………………………………………………………………i Abstract……………………………………………………………………………..iii 目錄…………………………………………………………………………………iv 第一章、緒論 Introduction………………………………………………………1 1.1 胰臟癌 (Pancreatic cancer) ……………………………………………1 1.2 長鏈非編碼RNA與癌症 (Long non-coding RNA and cancer) ………1 1.3 NEAT1…………………………………………………………………2 1.4 癌轉移 (cancer metastasis) ……………………………………………3 1.5 實驗目標…………………………………………………………………4 第二章、實驗材料及方法 Materials and methods………………………………5 2.1 細胞株與細胞培養 (Cell lines and culture conditions) ………………5 2.2 小干擾RNA轉染 (siRNA transfection) …………………………………5 2.3 核醣核酸萃取 (RNA isolation) …………………………………………6 2.4 即時定量反轉錄聚合酶連鎖反應(Real-time Quantitative reverse transcriptase PCR) ………………………………………………………7 2.5 蛋白質萃取 (Protein extraction) ………………………………………8 2.6 抗體 (Antibodies) ………………………………………………………8 2.7 西方墨點法與抗體 (Western blot and Antibodies)……………………8 2.8 傷口癒合爬行試驗 (Wound-healing migration assays)……………9 2.9 細胞侵襲能力試驗 (Cell invasion assays) ……………………………10 2.10 單細胞定位追蹤 (Single cell tracking) ………………………………10 2.11 細胞存活率試驗 (MTT cell proliferation assays)……………10 2.12 統計分析 (Statistical analyses) …………………………………………11 第三章、結果 Results………………………………………………………………12 3.1 利用RNA-seq預測可能與轉移相關的長鏈非編碼RNA：NEAT1…12 3.2 比較原發性細胞株(cell line derived from primary tumor)及轉移細胞株(cell line derived from metastatic site)NEAT1表現量差異………12 3.2.1 利用RT-qPCR驗證RNA-seq預測結果…………………………12 3.2.2 檢測CL1-0及CL1-5細胞株中NEAT1於mRNA層次表現的變化…13 3.3 分析抑制NEAT1表現與癌轉移(metastasis)的關係……………………13 3.3.1 在細胞株PK2以及CL1-5中抑制NEAT1的表現量使得細胞爬行 能力(migration ability)下降…………………………………………13 3.3.2 在細胞株PK2以及CL1-5中抑制NEAT1的表現量使得細胞侵襲 能力(invasion ability)下降…………………………………………15 3.3.3 在細胞株PK2以及CL1-5中抑制NEAT1的表現量對於EMT相 關基因表現的影響…………………………………………………15 3.4 其他細胞株-胰臟癌、大腸癌、肝癌中NEAT1的表現量與癌轉移的 關係………………………………………………………………………16 3.4.1檢測胰臟癌、大腸癌、肝癌細胞株中NEAT1的表現量…………16 3.4.2抑制NEAT1表現於細胞株AsPC-1、PL45以及HCT15會降低細 胞爬行能力…………………………………………………………17 3.4.3抑制NEAT1表現於細胞株AsPC-1、PL45以及HCT15對於EMT 相關基因表現的影響………………………………………………18 3.5 觀察瞬時轉染小干擾RNA(transient transfection of siRNA)抑制NEAT1對細胞生長之影響………………………………………………19 第四章、討論 Discussions…………………………………………………………20 第五章、實驗圖表 Figures and tables……………………………………………22 第六章、參考文獻 References……………………………………………………38 | |
| dc.language.iso | zh-TW | |
| dc.title | 抑制NEAT1表現量對癌轉移及癌侵襲能力之影響 | zh_TW |
| dc.title | NEAT1 Knockdown Attenuates Cancer Migration and Invasion | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳振都,田郁文 | |
| dc.subject.keyword | 胰臟癌,癌轉移,NEAT1, | zh_TW |
| dc.subject.keyword | pancreatic cancer,migration,NEAT1, | en |
| dc.relation.page | 41 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2015-02-24 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 病理學研究所 | zh_TW |
| 顯示於系所單位： | 病理學科所 | |
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