臺灣博碩士論文加值系統

組蛋白離胺酸去甲基酶KDM4A–D對於移除抑制性組蛋白離胺酸上的甲基團進而促進基因活化、癌細胞生長及癌症轉移扮演重要的表觀遺傳調控者。KDM4A與KDM4B經常過度表現於胃癌中。介白素-8 (IL-8)，一種促炎性趨化介素，也大量表現於胃癌中並與化療抗性及上皮間質轉化相關。值得注意的，受到病原體CagA陽性胃幽門螺蜁菌感染促使大量IL-8產生。然而，表觀遺傳調控者對於調控胃癌中胃幽門螺蜁菌引起的IL-8表現及致癌作用的機制仍不清楚。在本研究中，我們證實KDM4B顯著地激活IL-8產生，而非KDM4A/4C參與其中。細胞受到胃幽門螺蜁菌感染後則有更明顯地影響。西方墨點微陣列分析顯示JNK途徑對於KDM4B調控的IL-8產生是很重要的。此外，我們發現KDM4B藉由N端與c-Jun結合。染色質免疫沈澱分析顯示KDM4B與c-Jun共同調控IL-8、MMP1及ITGAV啟動子。IL-8啟動子活性分析顯示IL-8轉錄激活活性需要透過KDM4B去甲基化活性。與受到胃幽門螺蜁菌感染的AGS細胞相比，KDM4B的缺失導致透過胃幽門螺旋菌第四型分泌系統所需的整合素αV表現量下降及轉位的CagA下降進而減少IL-8產生及抑制創傷癒合能力。在胃癌中，高度KDM4B表現與c-Jun磷酸化程度連接到較差的臨床結果。綜合以上，我們的結果顯示KDM4B是c-Jun重要的表觀遺傳共活化者，一起共同參與胃幽門螺旋菌引起的反應。KDM4B可以作為一個具有潛力的胃癌治療標的。

Histone lysine demethylase 4 members (KDM4A–D) are important epigenetic regulators which remove repressive methyl marks from histone lysines to promote gene activation, cancer cell growth and metastasis. KDM4A and KDM4B are often overexpressed in gastric cancer. Interleukin-8 (IL-8), a pro-inflammatory chemokine, is also overexpressed in gastric cancer, which is correlated with chemo-resistance and epithelial-to-mesenchymal transition. Of note, infection with CagA-positive Helicobacter pylori, an etiologic agent of gastric cancer, promotes the production of IL-8. However, the role of the epigenetic regulator in modulating H. pylori-induced IL-8 expression and tumorigenesis in gastric cancer remains unclear. In this study, we report that KDM4B, rather than KDM4A/4C, significantly activates IL-8 production. An even more pronounced effect is seen in cells challenged with H. pylori. Micro-western analysis reveals that JNK pathway is important for KDM4B-mediated IL-8 production. Furthermore, we show that KDM4B interacts with c-Jun through its N-terminal domain. ChIP analysis reveals that KDM4B and c-Jun are co-recruited to the promoter region of IL-8, MMP1, and ITGAV. IL-8 promoter activity analysis reveals that the demethylase activity of KDM4B is essential in the IL-8 transactivation activity. As compared with H. pylori-infected AGS cells, the depletion of KDM4B downregulates the level of CagA translocation through integrin αV encoded by ITGAV, thereby reducing the level of IL-8 production and wound healing effect. The high levels of KDM4B and p-c-Jun are associated with a poorer clinical outcome in gastric cancer patients. Together, our results highlight that KDM4B is a crucial epigenetic co-activator of c-Jun, which cooperate together in response to H. pylori challenge. KDM4B thus serves as a potential therapeutic target in gastric cancer.

致謝......i
中文摘要......iii
Abstract......v
Abbreviation......vii
List of Figures......xi
List of Tables......xiii
Chapter 1. Introduction......1
1.1 The role of epigenetics in cancer development......1
1.2 The development and treatment of Gastric cancer......3
1.3 Gastric pathogen: Helicobacter pylori......5
1.4 Inflammatory response in tumor microenvironment......8
1.5 The objective of this study......9
Chapter 2. Materials and Methods......11
2.1 Cell lines and cell culture......11
2.2 Bacteria and culture......11
2.3 Antibodies and reagents......11
2.4 Cell line establishment......12
2.5 Human cytokine array......13
2.6 Quantitative real-time PCR (qRT-PCR)......13
2.7 Human IL-8 ELISA......13
2.8 Cell elongated hummingbird phenotype......14
2.9 Immunoblotting analysis......14
2.10 Immunoprecipitation (IP)......15
2.11 Luciferase activity assay......15
2.12 Chromatin Immunoprecipitation (ChIP) assay......16
2.13 Small interfering RNA (siRNA) transfection......17
2.14 Microarray......17
2.15 Micro-western array (MWA)......18
2.16 Wound healing assay......18
2.17 Immunohistochemistry (IHC)......18
2.18 Kaplan Meier-plotter (KM-plotter) survival analysis......19
2.19 Statistical analysis......20
Chapter 3. Results......21
3.1 KDM4B increases IL-8 production in H. pylori-infected gastric cancer cells.......21
3.2 KDM4B mediates the expression of AP-1- and NF-B-regulated genes......22
3.3 JNK/c-Jun pathway is important for KDM4B-regulated IL-8 production......23
3.4 KDM4B interacts with c-Jun and regulates the IL-8 promoter activity......24
3.5 KDM4B and c-Jun are co-recruited on IL-8 and MMP1 loci......27
3.6 KDM4B mediates the translocation of CagA......28
3.7 KDM4B regulates the expression of ITGAV encoding integrin V to promote the translocation of CagA......29
3.8 KDM4B and p-c-Jun are associated with poorer clinical outcome in gastric cancer......31
Chapter 4. Conclusion and Discussion......33
References......38
Figures and Tables......47

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