胃幽門螺旋菌 (Helicobacter pylori) 為一革蘭氏陰性、具螺旋狀的微需氧細菌。世界上超過 50 % 人口的胃部中有其存在。胃黏膜持續地感染胃幽門螺旋菌會引起慢性胃炎、胃潰瘍、十二指腸潰瘍，甚至是胃腺癌。由感染所引起相關的慢性發炎 (chronic inflammation) 會使胃上皮細胞損害，而持久性的上皮細胞損害可能會導致其不典型增生變化 (dysplastic changes) 及產生惡性變形，從長期來看，此惡性變化將可能進一步發展成胃癌形成。KDM4B 是一種具有 JmjC domain 的組蛋白去甲基酵素，可以針對組蛋白 H3 尾端鏈接三個甲基之第 9個及第 36個離氨酸 (H3K9me3及H3K36me3) 進行去甲基的反應。近期發現，在胃癌等許多癌症細胞中KDM4B具有過度表現之情形，但其與胃幽門螺旋菌感染的關係尚未可知。因此本研究之主要目的在於探討KDM4B是否會調控胃幽門螺旋菌所誘導之發炎反應，並聚焦於促進發炎反應之細胞激素 (proinflammatory cytokine)－ 白細胞介素-8 (Interleukin-8)的表現及其調控機制。本研究發現減少 KDM4B 的表現，會使胃癌細胞因胃幽門螺旋菌所誘導之白細胞介素-8的表現量下降。由冷光酶報告基因檢測 (Luciferase reporter assay)結果發現，減少 KDM4B 的表現，會降低轉染至胃癌細胞中白細胞介素-8之啟動子 (IL-8 promoter construct) 下游冷光酶活性。進一步轉染兩個不同之冷光酶報告基因質體－除去白細胞介素-8啟動子上的轉錄因子 AP-1 結合位 (AP-1 binding site-deleted construct, ΔAP-1) 及除去NF-κB結合位 (NF-κB binding site-deleted clone, ΔNF-κB) －發現，若除去白細胞介素-8啟動子上的轉錄因子 AP-1 結合位，也會降低其冷光酶活性。另一方面，微陣列分析結果顯示許多基因在控制組及 KDM4B 減少組細胞中具差異表現，而這些基因涵蓋了受刺激之反應、免疫系統過程、多生物過程 (multiorganism process) 、受傷害之反應、防禦反應以及對病毒之反應。經進一步分析，本研究發現因 KDM4B 減少且經胃幽門螺旋菌感染而降低表現量的基因中，與AP-1結合的基因佔全部之 21 % ，其中包含了白細胞介素-8。本研究也發現 KDM4B 與 c-Jun (AP-1組成之一) 能有物理性的結合。因此，由上述結果推測， KDM4B 可能會藉由與 AP-1 結合來調控白細胞介素-8及其他因胃幽門螺旋菌感染所誘導之基因的轉錄活性及表現。

Helicobacter pylori (H. pylori) is a gram-negative, spiral-shaped microaerophilic bacterium and inhabits the stomach of more than half of world’s population. Persistent H. pylori infection in human gastric mucosa causes chronic gastritis, gastric, duodenal ulcers, and even gastric adenocarcinoma. Enduring injury of epithelial cells caused by infection-associated chronic inflammation may lead to dysplastic changes, malignant transformation, which may further develop into gastric carcinogenesis in the long run. KDM4B [lysine (K)-specific demethylase 4]/JMJD2B (Jumonji C domain-containing 2) protein is a demethylase that targets histone H3 on lysine 9 and 36. Recently, the role of KDM4B in gastric carcinogenesis is reported. However, the relationship with H. pylori infection remains largely unknown. In this study, we characterized whether KDM4B regulated H. pylori-induced inflammation, focusing on the release of proinflammatory cytokine interleukin-8 (IL-8), and investigated the underlying mechanisms. Knocking down expression of KDM4B significantly reduced the release of IL-8 in H. pylori-infected AGS cells. Luciferase reporter assay showed knockdown of KDM4B decreased IL-8 promoter activity. Using different IL-8 promoter constructs [AP-1-NF-κB binding site-fused construct, AP-1 biding site-deleted construct (ΔAP-1), and NF-κB binding site-deleted construct (ΔNF-κB)] revealed that ΔAP-1–transfected cells had a significantly reduced level of luciferase activity. Microarray analysis revealed that differentially expressed genes between control and KDM4B-knockdown cells included those involved in response to stimulus, immune system process, multi-organism process, response to wounding, defense response and response to virus. Above all, 21% of down-regulated genes of KDM4B-knockdown with H. pylori infection were AP-1 binding genes including IL-8. Co-immunoprecipitation experiments showed that KDM4B was associated with c-Jun, a component of AP-1. Our results suggest that KDM4B might associate with AP-1 and co-regulate the transcriptional activity of IL-8 and other genes induced by H. pylori infection.

Contents
中文摘要 i
Abstract ii
致謝 iii
Contents………………………………………………………………………………………………v
List of Tables vii
List of Figures viii
Chapter 1 Introduction 1
1.1 Helicobacter pylori 1
1.2 H .pylori-induced pro-inflammatory response 1
1.3 H. pylori infection and gastric carcinogenesis 2
1.4 Posttranscriptional modifications and cancer 2
1.5 Histone modifications and histone demethylases 3
1.6 KDM4 family (KDM4A, KDM4B and KDM4C) and cancers 4
1.7 Inflammation and interleukin-8 (IL-8) 4
1.8 Transcriptional regulation of interleukin-8 (IL-8) 5
1.9 Goals and specific aims 6
Chapter 2 Materials and Methods 7
2.1 Cell lines and cell culture 7
2.2 KDM4A, KDM4B and KDM4C stable cell lines generation 7
2.2.1 Lentivirus production 7
2.2.2 Lentiviral infection and selection for KDM4A, KDM4B and KDM4C knockdown cell lines 8
2.3 H. pylori culture 8
2.4 H. pylori infection treatment 9
2.5 Protein extraction 9
2.6 Western blotting 10
2.7 Enzyme-linked immunosorbent assay (ELISA) for IL-8 10
2.8 RNA extraction and quantitative real-time PCR 11
2.8.1 RNA extraction 11
2.8.2 cDNA synthesis 12
2.8.3 Quantitative real-time PCR 12
2.9 Microarray analysis 12
2.10 Immunoprecipitation assay 13
2.11 Antibodies that were used in this study 13
2.12 Luciferase reporter assay 14
2.13 Statistical analysis 15
Chapter 3 Results 18
3.1 KDM4A, KDM4B and KDM4C were stably knocked down by shRNA lentiviral system respectively 18
3.2 IL-8 production was significantly reduced in KDM4B knockdown AGS cells 18
3.3 CagA contributed to H. pylori-induced IL-8 production that might be regulated by KDM4B 19
3.4 Knockdown of KDM4B decreased IL-8 promoter activity and different IL-8 promoter constructs revealed that ΔAP-1–transfected cells had significantly reduced activity 20
3.5 Characterization of the global expression pattern regulated by KDM4B 20
3.6 KDM4B was physically associated with c-Jun, a component of AP-1 23
Chapter 4 Discussion 23
Future aspect 27
References 28

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