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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 | 周綠蘋(Lu-Ping Chow) | |
dc.contributor.author | Ting-Yu Fan | en |
dc.contributor.author | 范婷羽 | zh_TW |
dc.date.accessioned | 2021-06-15T11:53:41Z | - |
dc.date.available | 2019-08-26 | |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-11 | |
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Posern, E-cadherin regulates MAL-SRF-mediated transcription in epithelial cells. J Cell Sci, 2010. 123(16): p. 2803-2809. 54. Yang, X., et al., Daxx, a novel Fas-binding protein that activates JNK and apoptosis. Cell, 1997. 89(7): p. 1067-1076. 55. Mongan, M., et al., Loss of MAP3K1 enhances proliferation and apoptosis during retinal development. Development, 2011. 138(18): p. 4001-4012. 56. Go, W.Y., et al., NFAT5/TonEBP mutant mice define osmotic stress as a critical feature of the lymphoid microenvironment. Proceedings of the National Academy of Sciences of the United States of America, 2004. 101(29): p. 10673-10678. 57. Kern, H.B., et al., Control of MicroRNA-21 expression in colorectal cancer cells by oncogenic epidermal growth factor/Ras signaling and Ets transcription factors. DNA and cell biology, 2012. 31(8): p. 1403-1411. 58. Iosub-Amir, A. and A. Friedler, Protein–protein interactions of ASPP2: an emerging therapeutic target. MedChemComm, 2014. 5(10): p. 1435-1443. 59. Trigiante, G. and X. Lu, ASPPs and cancer. Nature Reviews Cancer, 2006. 6(3): p. 217-226. 60. Rotem, S., et al., The structure and interactions of the proline-rich domain of ASPP2. Journal of biological chemistry, 2008. 283(27): p. 18990-18999. 61. Gorina, S. and N.P. Pavletich, Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2. Science, 1996. 274(5289): p. 1001. 62. Samuels-Lev, Y., et al., ASPP proteins specifically stimulate the apoptotic function of p53. Molecular cell, 2001. 8(4): p. 781-794. 63. Song, B., et al., Downregulation of ASPP2 in pancreatic cancer cells contributes to increased resistance to gemcitabine through autophagy activation. Molecular cancer, 2015. 14(1): p. 1. 64. Schittenhelm, M.M., et al., Attenuated Expression of A poptosis S timulating P rotein of p 53-2 (ASPP2) in Human Acute Leukemia Is Associated with Therapy Failure. PloS one, 2013. 8(11): p. e80193. 65. Mak, V.C., et al., Downregulation of ASPP2 in choriocarcinoma contributes to increased migratory potential through Src signaling pathway activation. Carcinogenesis, 2013: p. bgt161. 66. Meng, W.-D., et al., Helicobacter pylori infection and expressions of apoptosis-related proteins p53, ASPP2 and iASPP in gastric cancer and precancerous lesions. Pathologie Biologie, 2013. 61(5): p. 199-202. 67. Sottocornola, R., et al., ASPP2 binds Par-3 and controls the polarity and proliferation of neural progenitors during CNS development. Developmental cell, 2010. 19(1): p. 126-137. 68. Bergamaschi, D., et al., ASPP1 and ASPP2: common activators of p53 family members. Molecular and cellular biology, 2004. 24(3): p. 1341-1350. 69. Canning, P., F. von Delft, and A.N. Bullock, Structural basis for ASPP2 recognition by the tumor suppressor p73. Journal of molecular biology, 2012. 423(4): p. 515-527. 70. Naumovski, L. and M.L. Cleary, The p53-binding protein 53BP2 also interacts with Bc12 and impedes cell cycle progression at G2/M. Molecular and cellular biology, 1996. 16(7): p. 3884-3892. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49868 | - |
dc.description.abstract | 胃癌是全球十大癌症死因之一,在開發中國家較為常見。誘發胃癌產生的影響因子包含了宿主本身的免疫反應、生活環境與飲食習慣等,其中幽門螺旋桿菌 (Helicobacter pylori) 感染為最主要的原因。過去已有研究顯示幽門螺旋桿菌的感染確實與胃癌發生有關,然而其詳細的致癌機轉仍不清楚。
微小核醣核酸 (miRNAs) 為具有約22個核甘酸的小分子RNA,能藉由與特定mRNA基因的3端不轉譯區 (3’UTR) 結合,抑制mRNA轉譯或造成其裂解,進而調控相關的基因表現。近年來已有報導指出幽門螺旋桿菌感染後,會使細胞內miRNAs有異常的表現,其中miR-21為已知的致癌miRNA (oncomir),在許多癌症中都有大量表現的情形,並可藉由抑制其下游基因表現導致癌症產生,因此探討miR-21調控的下游目標基因有助於了解胃癌的發生。 為了釐清miR-21是藉由調控哪些目標基因影響胃腺癌上皮細胞AGS的功能,本實驗室利用了SILAC定量質譜學方法與預測miRNAs目標基因的資料庫miRsystem找到了6個miR-21的潛力目標,分別為DAXX、MAP3K1、NFAT5、PDCD4、RASA1及ASPP2。這6個潛力目標中,我們發現ASPP2 mRNA表現量在miR-21大量表現的細胞中下降情形最為顯著,並且也觀察到大量表現miR-21會減少ASPP2蛋白表現,因此讓我們對ASPP2產生了興趣。我們利用報導基因分析 (luciferase report assay) 證實ASPP2的確為miR-21的目標基因,同時也利用慢病毒攜帶shRNA方法抑制AGS細胞中ASPP2的表現,觀察其參與之功能。實驗結果發現抑制ASPP2表現量會增強細胞增生能力與抑制細胞凋亡,並且也會增加細胞貼附性與非貼附性群落形成的能力。進一步我們利用免疫組織化學染色觀察到人類胃癌組織中ASPP2的表現量低於在胃炎組織的表現。因此我們提出一結論,高度表現的miR-21會抑制其下游目標基因ASPP2的表現,進而導致胃癌的發生。 | zh_TW |
dc.description.abstract | Gastric cancer (GC) is one of the leading causes of cancer death in the world and more common in developing countries. The interaction of host, bacteria, and environmental factors involve in the development of gastric cancer. Among these, Helicobacter pylori infection is the most common risk factor of GC. Although the role of H. pylori in gastroduodenal diseases has been proposed, the detailed molecular pathway remains unclear.
MicroRNAs (miRNAs) are small approximately 22-nucleotide RNAs that modulate gene expression via binding to the 3’ untranslated region (3’UTR) of target mRNAs resulting in translational repression or degradation. Recent studies have suggested that some miRNAs expression is dysregulated in human gastric cells by H. pylori infection. Among these, miR-21 is a well-known oncomir which is upregulated in many cancers and leads to tumorigenesis by regulating its downstream target genes. Therefore, investigating downstream target genes of miR-21 could help us to understand the incidence of gastric cancer. In order to clarify how miR-21 affects the function of AGS cells, we used SILAC-based quantitative MS and miRsystem, an integrated system for predicting of miRNA targets, to find the potential target genes of miR-21. Finally, we found 6 potential targets, including DAXX, MAP3K1, NFAT5, PDCD4, RASA1, and ASPP2. Among 6 potential targets, we observed that the expression of ASPP2 mRNA was decreased most significantly in miR-21-overexpressed cells. Overexpression of miR-21 also decreased the expression of ASPP2 protein. Then we also found that miR-21 could bind to the 3’UTR of ASPP2, suggesting that ASPP2 is a direct target of miR-21. In order to realize the function of ASPP2, we knockdown of ASPP2 in AGS by using lentivirus-mediated shRNA. The results showed that knockdown of ASPP2 increased cell proliferation and decreased 5-Fu-induced apoptosis in AGS. Knockdown of ASPP2 also increased anchor-dependent and anchor-independent colony-forming ability of AGS cells. Furthermore, we found that the expression of ASSP2 was lower in human gastric cancer tissue than in gastritis tissue. As a result, we concluded that ASPP2 is down-regulated by miR-21 overexpression and may lead to gastric carcinogenesis. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T11:53:41Z (GMT). No. of bitstreams: 1 ntu-105-R03442017-1.pdf: 3642027 bytes, checksum: 4f7b10eab9f075258c38b31ab155ee08 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 口試委員會審定書 I
謝誌 II 中文摘要 III Abstract IV 縮寫 VI 第一章 導論 1 第一節 胃癌 1 1.1 胃癌的流行病學及其分類 1 1.2 胃癌危險因子 (risk factors) 1 第二節 幽門螺旋桿菌 3 2.1 幽門螺旋桿菌的型態、特徵及流行病學調查 3 2.2 幽門螺旋桿菌的致癌訊息傳遞路徑 (oncogenic pathways) 4 第三節 幽門螺旋桿菌與微小核醣核酸 (miRNAs) 的關係 5 3.1 微小核醣核酸的生合成路徑及其功能 5 3.2 幽門螺旋桿菌感染引起微小核醣核酸異常表現 7 第四節 微小核醣核酸21 (miR-21) 8 4.1 微小核醣核酸21所扮演的致癌角色 (oncomir) 8 4.2 微小核醣核酸21與幽門螺旋桿菌及胃癌的關係 8 4.3 微小核醣核酸21在胃癌中已知的下游目標基因 9 第五節 研究動機 11 第六節 研究策略 12 第二章 實驗材料 13 第一節 胃腺癌上皮細胞 13 第二節 組織切片 13 第三節 儀器及裝置 13 第四節 質體 14 第五節 酵素 14 第六節 抗體 14 第七節 試劑組及藥品 15 第八節 軟體與資料庫 17 第三章 實驗方法 18 第一節 胃腺癌上皮細胞的培養 18 1.1 培養基 (medium) 的配置 18 1.2 細胞培養 (cell culture) 18 1.3 細胞計數 18 第二節 利用重組腺病毒大量表現miR-21 18 2.1帶有miR-21基因之重組腺病毒載體製作 18 2.2重組腺病毒生產 19 2.3大量表現miR-21細胞株的建立 20 第三節 反轉錄聚合酶鏈鎖反應 (RT-PCR) 20 3.1 細胞RNA萃取 20 3.2 miRNAs與一般基因mRNA反轉錄反應 (reverse transcription, RT) 21 3.3聚合酶鏈鎖反應與瓊脂凝膠電泳((PCR and agarose gel electrophoresis) 22 3.4即時定量聚合酶鏈鎖反應 (real-time quantitative PCR, qPCR) 23 第四節 蛋白質分析法 23 4.1 細胞蛋白質萃取 23 4.2 蛋白質濃度測定 (BCA protein assay) 24 4.3 十二烷基硫酸鈉-聚丙烯醯胺膠體電泳分析 (SDS-PAGE) 24 4.4 西方墨點法 (western blotting) 26 第五節 小髮夾RNA (shRNA) 抑制目標蛋白 27 5.1 菌株培養 27 5.2 含有shRNA之中量質體DNA萃取 27 5.3 含有shRNA之慢病毒(lentivirus)製備 29 5.4 以含有shRNA之慢病毒(lentivirus)感染胃腺癌上皮細胞與抗生素篩選 29 5.5穩定抑制目標蛋白細胞株(stable clone)的建立 30 第六節 細胞生存能力試驗 (MTT asaay) 30 6.1 MTT reagent的配置 30 6.2 細胞準備 30 6.3 MTT活性偵測 31 第七節 細胞凋亡實驗分析 (Apoptosis assay) 31 7.1 細胞準備 31 7.2 利用西方墨點法 (western blotting) 觀察細胞凋亡相關蛋白的變化 31 第八節 免疫組織化學染色 (Immunohistochemistry, IHC) 32 第四章 實驗結果 33 第一節 利用線上預測軟體分析miR-21與潛力目標mRNA 3’ UTR結合位置 33 第二節 大量表現miR-21對潛力目標mRNA與蛋白表現量的影響 34 2.1 建立miR-21大量表現的細胞株 34 2.2 觀察miR-21大量表現對潛力目標mRNA表現量改變的情形 34 第三節 大量表現miR-21對ASPP2蛋白表現量的影響 35 第四節 確認miR-21的目標基因ASPP2 35 第五節 利用基因抑制 (gene knockdown) 方式分析ASPP2的細胞功能性 36 5.1 建立穩定抑制ASPP2表現量的胃腺癌上皮細胞株 37 5.2 抑制ASPP2表現量對細胞增生能力(cell proliferation)的影響 37 5.3 抑制ASPP2表現量對細胞凋亡(cell apoptosis)的影響 37 5.4 抑制ASPP2表現量對細胞群落形成能力(cell colony formation)的影響 38 5.5抑制ASPP2表現量對上皮細胞間質轉化 (epitheilial-to-mesenchymal transition, EMT) 相關指標蛋白表現量的影響 38 第六節 ASPP2於人類胃發炎與胃癌組織的表現量 39 第五章 討論 40 第一節 實驗方法學討論 40 第二節 微小核醣核酸21與其目標基因對胃癌的影響 40 第三節 ASPP2與微小核醣核酸21之間在胃癌的關聯性 41 3.1 ASPP2之結構與功能 41 3.2 ASPP2與細胞增生 (proliferation) 的關係 42 3.3 ASPP2與細胞凋亡 (apoptosis) 的關係 42 3.4 ASPP2與細胞轉移 (metastasis) 的關係 42 3.5 ASPP2於胃部臨床組織的表現 43 第四節 結語與未來展望 44 4.1 幽門螺旋桿菌致癌機轉與miR-21及其目標蛋白ASPP2之間的關聯性 44 4.2 未來工作與研究方向 45 第六章 參考文獻 46 圖表 51 附錄 61 | |
dc.language.iso | zh-TW | |
dc.title | 找尋並探討胃腺癌上皮細胞中微小核醣核酸21之可能標的蛋白 | zh_TW |
dc.title | To discover and characterize potential miR-21 targets in gastric adenocarcinoma cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林中梧(Chung-Wu Lin),楊智欽(Jyh-Chin Yang) | |
dc.subject.keyword | 胃癌,幽門螺旋桿菌,微小核醣核酸21,ASPP2, | zh_TW |
dc.subject.keyword | Gastric cancer,Helicobacter pylori,miR-21,ASPP2, | en |
dc.relation.page | 72 | |
dc.identifier.doi | 10.6342/NTU201602338 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-11 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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