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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 周綠蘋 | zh_TW |
dc.contributor.advisor | Lu-Ping Chow | en |
dc.contributor.author | 黃伯仕 | zh_TW |
dc.contributor.author | Bo-Shih Huang | en |
dc.date.accessioned | 2023-09-26T16:20:03Z | - |
dc.date.available | 2023-11-09 | - |
dc.date.copyright | 2023-09-26 | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2023-08-05 | - |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90338 | - |
dc.description.abstract | 幽門螺旋菌 (H. pylori) 感染會影響宿主細胞的存活途徑,包括細胞凋亡和增殖,而其失衡是H. pylori誘導胃癌 (HPGC) 發展的關鍵。H. pylori感染可誘導microRNAs表達的變化,其可能涉及GC的發展。生物資訊學分析顯示,在HPGC中microRNA-21(miR-21)表現顯著上升。此外,定量蛋白質體學和in silico預測被用於識別miR-21的潛在標的。透過功能富集和群集相互作用網絡分析,我們在與細胞死亡和存活,細胞運動以及細胞生長和增殖相關的三個功能簇中發現了miR-21標的之五個候選蛋白,PDCD4,ASPP2,DAXX,PIK3R1和MAP3K1。 H. pylori誘導的miR-21過度表達抑制ASPP2。此外,在HPGC腫瘤組織中,ASPP2與miR-21表現呈現負相關。總之,ASPP2在HPGC中被識別為miR-21的標的。在此,我們使用凋亡試驗觀察到H. pylori誘導的ASPP2抑制增強了GC細胞之抗凋亡能力。使用蛋白質相互作用網絡和免疫共沉澱實驗,我們發現了CHOP作為H. pylori感染的GC細胞中ASPP2促凋亡活性的直接介質。從機制上講,ASPP2抑制促進p300介導的CHOP降解,進而抑制CHOP介導的Noxa,Bak的轉錄和Bcl-2的抑制,進而在H. pylori感染之GC細胞中實現抗凋亡。臨床病理學分析顯示了ASPP2表達下降與HPGC風險增加和預後不良之間的相關性。綜合以上,我們發現H. pylori透過誘導miR-21介導的ASPP2 / CHOP介導信號之抑制來導致抗凋亡作用,此發現對於開發HPGC管理和治療策略提供了一個新的視角。 | zh_TW |
dc.description.abstract | Helicobacter pylori (H. pylori) infection affects cell survival pathways, including apoptosis and proliferation in host cells, and disruption of this balance is the key event in the development of H. pylori-induced gastric cancer (HPGC). H. pylori infection induces alterations in microRNAs (miRNAs) expression that may be involved in GC development. Bioinformatic analysis showed that microRNA-21 (miR-21) is significantly upregulated in HPGC. Furthermore, quantitative proteomics and in silico prediction were employed to identify potential targets of miR-21. Following functional enrichment and clustered interaction network analyses, five candidates of miR-21 targets, PDCD4, ASPP2, DAXX, PIK3R1, and MAP3K1, were found across three functional clusters associated with cell death and survival, cellular movement, and cellular growth and proliferation. ASPP2 is inhibited by H. pylori-induced miR-21 overexpression. Moreover, ASPP2 is inversely correlated with miR-21 levels in HPGC tumor tissues. Taken together, ASPP2 was identified as a miR-21 target in HPGC. Here, we observed H. pylori-induced ASPP2 suppression enhances resistance to apoptosis in GC cells using apoptosis assays. Using protein interaction network and co-immunoprecipitation assay, we identified CHOP as a direct mediator of the ASPP2 pro-apoptotic activity in H. pylori-infected GC cells. Mechanistically, ASPP2 suppression promotes p300-mediated CHOP degradation, which in turn inhibits CHOP-mediated transcription of Noxa, Bak, and suppression of Bcl-2, leading to anti-apoptotic effects in GC cells after H. pylori infection. Clinicopathological analysis revealed correlations between decreased ASPP2 expression and higher HPGC risk and poor prognosis. In summary, the discovery of H. pylori-induced anti-apoptosis via miR-21-mediated suppression of ASPP2/CHOP-mediated signaling provides a novel perspective on developing HPGC management and treatment. | en |
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dc.description.tableofcontents | 誌謝 iii
摘要 iv Abstract v Abbreviations vii Table of Contents 1 Chapter I – Overview and Rationale 5 1.1 H. pylori Microbiology 5 1.2 H. pylori infection and gastric diseases 5 1.2.1 gastritis 5 1.2.2 peptic ulcers 6 1.2.3 MALT lymphoma 6 1.2.4 gastric cancer 7 1.3 H. pylori virulence factors 7 1.3.1 urease 7 1.3.2 flagella 7 1.3.3 adhesion factors 8 1.3.4 lipopolysaccharide (LPS) 8 1.3.5 CagA 8 1.3.6 VacA 9 1.4 H. pylori-induced host responses 9 1.4.1 H. pylori-induced oncogenic proteins 9 1.4.2 H. pylori-induced oncogenic miRNAs 10 Chapter II – Study of the Mechanism and Clinical Relevance of miR-21 target ASPP2 in H. pylori-Induced Gastric Cancer 11 2.1 Introduction 11 2.1.1 oncogenic role of miR-21 via targeting ASPP2 11 2.1.2 tumor suppressive role of ASPP2 via promoting CHOP-mediated apoptotic pathway 11 2.1.3 prognostic value of ASPP2 in cancers 12 2.2 Materials and Methods 13 2.2.1 human subjects and tissue specimens 13 2.2.2 bioinformatic analysis 13 2.2.3 in situ hybridization and immunohistochemistry (IHC) 14 2.2.4 cell culture, H. pylori culture and infection 15 2.2.5 vector construction, transfection and viral transduction 16 2.2.6 SILAC-based quantitative proteomic analysis 17 2.2.7 qPCR, chromatin immunoprecipitation (chIP) assays and luciferase reporter assays 17 2.2.8 immunoblotting and co-immunoprecipitation (co-IP) assays 18 2.2.9 cell proliferation, wound healing and invasion assays 18 2.2.10 terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and annexin V/ propidium Iodide (PI) staining apoptosis assays 19 2.2.11 statistical analysis 19 2.3 Results 20 2.4 Discussion 30 Chapter III – Conclusion and Perspectives 35 List of Tables 38 Table 1. Demographic and clinicopathological features and ASPP2 levels in HPGC, HPIM, HPGS and HP (-) healthy control groups 39 Table 2. ORs of demographic features and ASPP2 levels for HPGC determined via logistic regression analysis 41 Table 3. HRs of demographic and clinicopathological features and ASPP2 expression in HPGC patients determined via Cox proportional hazards analysis 43 List of Figures 46 Figure 1. MiR-21 is identified as a potentially upregulated miRNA in HPGC. 47 Figure 2. MiR-21 overexpression in HPGC tissues 48 Figure 3. H. pylori induces NF-B-mediated miR-21 overexpression in GES-1 cells. 49 Figure 4. Upregulation of miR-21 impacts proliferation, migration, and invasion of GES-1 cells. 51 Figure 5. Quantitative proteomics reveals altered functional proteome in GC cells after miR-21 overexpression. 53 Figure 6. H. pylori-induced ASPP2 suppression is mediated by miR-21 in GC cells. 54 Figure 7. ASPP2 downregulation and negative correlation with miR-21 levels in HPGC tissues 55 Figure 8. ASPP2 plays a role in AGS cell apoptosis. 56 Figure 9. ASPP2 plays a role in MKN45 cell apoptosis. 57 Figure 10. The effect of H. pylori infection on apoptosis in MKN45 cells 58 Figure 11. H. pylori induces ASPP2 downregulation and inhibition of apoptosis in AGS cells. 59 Figure 12. H. pylori induces ASPP2 downregulation and inhibition of apoptosis in MKN45 cells. 60 Figure 13. Cleaved caspase 3 downregulation and positive correlation with ASPP2 suppression in HPGC tissues 61 Figure 14. Identification of CHOP as a potential mediator of ASPP2-mediated apoptosis 62 Figure 15. ASPP2 suppression promotes p300-mediated CHOP degradation in H. pylori-infected AGS cells. 63 Figure 16. CHOP regulates Noxa, Bak, and Bcl-2 expression in H. pylori-infected AGS cells. 65 Figure 17. ASPP2 suppression inhibits CHOP-mediated apoptotic signaling in H. pylori-infected AGS cells. 66 Figure 18. H. pylori-induced ASPP2 suppression inhibits CHOP-mediated apoptotic signaling in MKN45 cells. 67 Figure 19. H. pylori-induced ASPP2 suppression promotes p300-mediated CHOP degradation in AGS cells. 68 Figure 20. H. pylori-induced ASPP2 suppression promotes p300-mediated CHOP degradation in MKN45 cells. 69 Figure 21. MiR-21 is involved in H. pylori-induced ASPP2 suppression to inhibit CHOP-mediated regulation of Noxa, Bak and Bcl-2. 71 Figure 22. MiR-21 overexpression and downregulated ASPP2 and cleaved caspase 3 in HPGC and HPIM relative to HPGS tissues 73 Figure 23. Decreased ASPP2 expression is associated with poor prognosis in HPGC. 75 Figure 24. H. pylori-induced miR21-mediated ASPP2 suppression confers resistance to apoptosis through inhibition of ASPP2/CHOP-mediated transcription of Noxa and Bak and the suppression of Bcl-2 in GC cells. 76 References 77 Supplementary Tables 87 Supplementary Table 1. List of primer sequences, small nucleotide sequences and target sequences of siRNA or shRNA 88 Supplementary Table 2. List of antibodies used in this study 93 Supplementary Table 3. Identification of 47 downregulated proteins (fold change < 0.77) in AdmiR-21 AGS cells by both SILAC and miRsystem approaches 94 Supplementary Table 4. Enriched biological functions of 47 downregulated (fold change<0.7) proteins in AdmiR-21 AGS cells via IPA 98 Supplementary Figures 99 Supplementary Figure 1. H. pylori induces NF-B-mediated miR-21 overexpression in AGS cells. 100 Supplementary Figure 2. Upregulation of miR-21 impacts proliferation, migration, and invasion of AGS cells. 103 Supplementary Figure 3. Strategic flowchart for the identification of 47 downregulated candidate targets of miR-21 in AGS cells 104 Supplementary Figure 4. Identification of potential miR-21 targets in GC cells 105 Supplementary Figure 5. Representative quantitative mass spectrum of potential miR-21 target, ASPP2 106 Supplementary Figure 6. Modulation of ASPP2 by miR-21 in AGS cells. 107 Supplementary Figure 7. ASPP2 as a direct miR-21 target in AGS cells. 108 Supplementary Figure 8. The effect of H. pylori infection on apoptosis in AGS cells 109 Supplementary Figure 9. CHOP plays role in apoptosis in AGS cells. 110 Supplementary Figure 10. H. pylori-induced ASPP2 suppression inhibits CHOP-mediated apoptotic signaling in AGS cells. 111 | - |
dc.language.iso | zh_TW | - |
dc.title | miR-21標的蛋白ASPP2其調控幽門螺旋桿菌誘導胃之機制與臨床相關性 | zh_TW |
dc.title | The Mechanism and Clinical Relevance of miR-21 target ASPP2 in Helicobacter pylori-Induced Gastric Cancer | en |
dc.type | Thesis | - |
dc.date.schoolyear | 111-2 | - |
dc.description.degree | 博士 | - |
dc.contributor.oralexamcommittee | 吳明賢;傅化文;顏伯勳;高正彥 | zh_TW |
dc.contributor.oralexamcommittee | Ming-Shiang Wu;Hua-Wen Fu;Bo-Shiun Yan;Cheng-Yen Kao | en |
dc.subject.keyword | 幽門螺旋桿菌,胃癌,miR-21,ASPP2,抗凋亡, | zh_TW |
dc.subject.keyword | H. pylori,gastric cancer,miR-21,ASPP2,anti-apoptosis, | en |
dc.relation.page | 112 | - |
dc.identifier.doi | 10.6342/NTU202303084 | - |
dc.rights.note | 未授權 | - |
dc.date.accepted | 2023-08-07 | - |
dc.contributor.author-college | 醫學院 | - |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | - |
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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