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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 阮雪芬 | |
| dc.contributor.author | Sheng-Chueh Tsai | en |
| dc.contributor.author | 蔡聖爵 | zh_TW |
| dc.date.accessioned | 2021-06-17T00:23:46Z | - |
| dc.date.available | 2013-06-27 | |
| dc.date.copyright | 2012-06-27 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2012-05-18 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66155 | - |
| dc.description.abstract | 微型核醣核酸是一種長約22鹼基的小片段內生型核醣核酸，其本身不會轉譯出蛋白質，但可藉由直接結合標靶訊息核醣核酸 (mRNA)的 3’-非轉譯區 (3‘UTR)，抑制蛋白質的轉譯或是造成mRNA的降解，進而負調控基因的表現。近年來發現微型核醣核酸不只對生物的發育有重要的影響，同時在基因表現的平衡中扮演重要的角色。最近的研究更指出微型核醣核酸對於癌症的發生、發展與遷移能力有密切的關係。我們將微型核醣核酸-148a (miR-148a)轉染至胃癌細胞株AGS並觀察其生長狀態以及細胞遷移能力的表現，結果顯示有轉染miR-148a的細胞其生長有明顯的減緩，遷移能力也相對低於對照組。進一步使用同位素標記絕對與相對定量 (iTRAQ)、網路分析軟體 (Ingenuity Pathway Analysis)、網路預測微型核醣核酸標靶基因軟體 (Targetscan 5.1)以及蛋白質網路建構資料庫STRING 8.3分析miR-148a對AGS細胞蛋白質層次的調控，並建構出可能的調控網路。結果發現，有四個受到負調控的蛋白質參與在AKT訊息傳遞路徑上。另外有一個預測為標靶基因的蛋白質FXR1其蛋白質表現量下降。根據前人的研究報導指出， FXR1表現量下降可導致TNF-α表現量上升，使得TNF-α促進細胞凋亡(apoptosis)。根據我們的實驗結果與建構之蛋白質調控網路認為，miR-148a可能主要透過抑制AKT訊息傳遞路徑上的蛋白質表現，而降低細胞的存活能力，另外也可能直接抑制FXR1的表現進而促進細胞的死亡。
另外，本實驗室以SAM分析GEO上胃癌組織與正常胃部細胞的資料，發現MTA2的RNA在胃癌組織中的表現為正常細胞中的3.67倍。實驗結果顯示TSGH細胞株內生miR-148a的量是AGS細胞株的2.91倍，大量轉染miR-148a入TSGH細胞株會導致MTA2的RNA表現量下降約20%，但是在冷光酶活性測定分析結果表示，MTA2並非miR-148a的標靶基因，最後以西方墨點法測得MTA2蛋白質表現量顯示下降了0.93倍。此實驗結果顯示，MTA2受到miR-148a的表現在RNA以及蛋白質層次上受到抑制，可能進而導致TSGH細胞的生長以及遷移能力。 | zh_TW |
| dc.description.abstract | MicroRNAs (miRNAs) are endogenous small non-coding RNA about 22 nucleotides in length. They can directly bind to target genes, causing down-regulation of their expression post-transcriptionally by suppressing protein translation or degrading mRNA. In this study, we found a tumor suppressor miRNA, miR-148a, could decrease cell growth and metastasis of gastric cancer cells. To further understand the regulatory mechanism of miR-148a in tumor progression of gastric cancer, we used isobaric tags for relative and absolute quantitation (iTRAQ) method to identify differentially expressed proteins in miR-148a-over-expressed AGS cells and construct miR-148a-regulated network. We further analyzed the enriched functions within its network using Ingenuity Pathway Analysis (IPA), STRING 8.3 and TargetScan 5.1 database. Our results revealed that the most enriched biological process was AKT signaling, known as a cell survival-related pathway. We also identified many down-regulated proteins involved in AKT signaling, including Bcl-2-like 1 protein (BCL2L1), Heme oxygenase-1(HMOX1), Integring-linked kinase (ILK) and Glycogen synthase kinase-3 alpha (GSK3A), based on iTRAQ analysis. On the other hand, we identified a predicted target of miR-148a, Fragile X-related protein (FXR1), was down-regulated in miR-148a over-expressed tumor cells. Study reports that down-regulates FXR1 could increase TNFα expression, and cause apoptosis through TNFα. Taken together, these results suggest that miR-148a may suppress cell survival through regulating FXR1-AKT network.
We analyzed microarray data from GEO by using SAM. The RNA expression of MTA2 in tumor tissue is 3.67 times higher than normal tissue. Furthermore, the endogenesis RNA expression of miR-148a in TSGH is 2.91 times higher than in AGS. As we transfect miR-148a precursor into TSGH, the RNA expression of MTA2 is downregulated up to 20%, but luciferase assay data revealed that MTA2 RNA is not the direct target of miR-148a. For further examination, the protein level of MTA2 is decreased 0.93 times under overexpression of miR-148a. MiR-148a might effect the expression of MTA2 in RNA and protein level, and regulate growth and migration ability of TSGH. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T00:23:46Z (GMT). No. of bitstreams: 1 ntu-100-R98b43032-1.pdf: 1596407 bytes, checksum: 6851c5aac74946af966e6a16af674cd0 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 誌謝 ............................................................................................................................... i
中文摘要 .....................................................................................................................iii Abstract ....................................................................................................................... iv 第一章 前言與背景介紹 ........................................................................................... 1 1-1 微型核醣核酸 (miRNA) .................................................................................. 1 1-1-1 微型核醣核酸 (miRNA)的介紹 ............................................................... 1 1-1-2 微型核醣核酸與小型干擾核醣核酸 (small interfering RNA, siRNA)的差異 ............................................................................................................. 2 1-1-3 微型核醣核酸在癌症的角色 .................................................................... 2 1-2 胃癌(Gastric cancer)簡介 ................................................................................ 3 1-3 AKT 訊息傳遞路徑介紹 ................................................................................ 4 1-4 IPA (Ingenuity Pathway Analysis)的介紹........................................................ 4 1-5 STRING (Search Tool for the Retrieval of Interacting Genes/Proteins)的介紹 5 1-6 DAVID (Database of Annotation, Visualization and Integrated Discovery)...... 5 1-7 MTA2 (Metastasis associated family 1, member 2)的介紹 .............................. 6 第二章 實驗材料與方法 ............................................................................................. 7 2-1 STRING的操作 ................................................................................................ 7 2-2 DAVID的操作 ................................................................................................. 7 2-3 細胞培養 ........................................................................................................... 8 2-3-1 解凍增殖 ................................................................................................. 8 2-3-2 細胞繼代 ................................................................................................. 8 2-3-3 細胞計數 ................................................................................................. 9 2-4 轉染作用 ........................................................................................................... 9 2-5 細胞總核醣核酸萃取 ..................................................................................... 10 2-6 反轉錄總量RNA反應( Total RNA Reverse transcription) ............................. 10 2-7 反轉錄微型核醣核酸反應(miRNA Reverse Transcription) ............................ 11 2-8 SYBG即時定量聚合酶連鎖反應轉 (Real-time PCR) ................................... 12 2-9 Taqman即時定量聚合酶連鎖反應轉 (Real-time PCR)................................. 13 2-10 pMIR-REPORT-MTA2 3‘UTR expression vector質體構築 ......................... 13 2-10-1 聚合酶連鎖反應 (Polymerase Chain reaction, PCR) .......................... 13 2-10-2 2%洋菜膠體製備與電泳分析 ............................................................. 14 2-10-3 膠體萃取DNA (Bioman EasyPrue PCR/Gel Extraction kit) .......... 15 2-10-4 DNA限制酶反應 ................................................................................ 15 2-10-5 接合作用 (Ligation)............................................................................ 16 2-10-6 轉型作用與藍白篩選 .......................................................................... 16 2-11 冷光酶活性測定分析 (Luciferase reporter assay) ........................................ 17 2-11-1 轉染作用 ............................................................................................... 17 2-11-2 Luciferase與 β-galactosidase 訊號偵測 .............................................. 17 2-12 Bradford 蛋白質定量 ................................................................................... 18 2-13 西方墨點法 (Western Blot) .......................................................................... 19 第三章 結果與討論 ................................................................................................. 21 3-1 以IPA和DAVID分析iTRAQ的結果 ......................................................... 21 3-2 以STRING建構PTEN和AKT與受miR-148a負調控之蛋白質間的網路 22 3-3 AKT訊息傳遞路徑與iTRAQ結果之間的關係 ............................................ 22 3-4 ILK與AKT之間的關係及其對細胞遷移之影響 ......................................... 23 3-5 GSK3-α對細胞生長之影響 ........................................................................... 24 3-6 HMOX1與AKT之間的關係及其對細胞 ..................................................... 24 3-7 BCL2L1與AKT之間的關係及其對細胞 ..................................................... 25 3-8 比對Targetscan以及miRanda預測之miR-148a標靶基因與iTRAQ結果 . 26 3-9 FXR1被預測為miR-148a的標靶基因 .......................................................... 26 3-10 討論以DAVID分析Targetscan預測基因與IPA分析的iTRAQ結果之間的異同 ............................................................................................................ 27 3-11 MTA2可能影響癌細胞的生長與遷移作用 ................................................. 27 3-12 以real-time PCR確認miR-148a在胃癌細胞TSGH與AGS中的含量 ..... 28 3-13 以real-time PCR確認轉染miR-148a的效率 .............................................. 28 3-14 以real-time PCR確認轉染miR-148a後MTA2 RNA量的改變 ................. 29 3-15 以Luciferase Assay確認MTA2的3‘UTR是否為miR-148a的直接目標 . 29 3-16 以西方墨點法確認miR-148a是否影響MTA2在TSGH中的表現 ........... 30 第四章 結論 ............................................................................................................ 32 第五章 圖 ................................................................................................................ 33 第六章 表 ................................................................................................................ 44 參考文獻 .................................................................................................................... 53 | |
| dc.language.iso | zh-TW | |
| dc.subject | MTA2 | zh_TW |
| dc.subject | 微型核醣核酸 | zh_TW |
| dc.subject | 胃癌 | zh_TW |
| dc.subject | iTRAQ | zh_TW |
| dc.subject | IPA | zh_TW |
| dc.subject | PI3K/AKT訊息傳遞路徑 | zh_TW |
| dc.subject | gastric cancer | en |
| dc.subject | PI3K/AKT signaling | en |
| dc.subject | IPA | en |
| dc.subject | iTRAQ | en |
| dc.subject | miRNA | en |
| dc.title | 探討微型核醣核酸miR-148a於胃癌細胞的蛋白質調控網路 | zh_TW |
| dc.title | MiR-148a-regulated Protein Networks in Human
Gastric Cancer Cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃宣誠,徐駿森 | |
| dc.subject.keyword | 微型核醣核酸,胃癌,iTRAQ,IPA,PI3K/AKT訊息傳遞路徑,MTA2, | zh_TW |
| dc.subject.keyword | miRNA,gastric cancer,iTRAQ,IPA,PI3K/AKT signaling, | en |
| dc.relation.page | 58 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-05-18 | |
| 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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