臺灣博碩士論文加值系統

胃癌是目前全世界第二致死的癌症。抑癌基因的表基因默化已成為胃癌發生的一個重要的基礎機制。過去的研究顯示，在胃癌中，幽門螺旋桿菌的感染造成活化JAK/STAT3訊息傳遞。然而，對於此訊息傳遞的異常活化的機制到目前為止尚未完全明白。因此我們假設，胃癌中JAK/STAT的訊息傳遞的異常活化會導致STAT3的標的基因受到表基因的調控而沉默。在我們過去的研究中，我們已經證明，在JAK/STAT訊息傳遞沒有持續活化的MKN28細胞中，持續活化的STAT3使其下游標的基因：NR4A3和LOXL4，透過啟動子CpG島甲基化而造成表基因沉默。將NR4A3送入JAK/STAT訊息傳遞持續活化的AGS細胞中，NR4A3的表達使AGS細胞的細胞群落形成分析 (colony formation assay) 以及軟瓊脂分析 (soft agar assay) 的細胞群落減少，還有細胞增殖率變慢。且在臨床檢體方面，我們利用定量性甲基性特異性連鎖聚合酶反應分析 (quantitative methylated-specific PCR, qMSP) 後發現，NR4A3的甲基化跟胃癌腫瘤檢體的STAT3活化有正相關，且病人的甲基化程度跟病人的存活率成反比。但是，與其結果相反，腸上皮化生 (intestinal metaplasia, IM) 病人的LOXL4甲基化比胃癌與非腫瘤胃炎檢體比起來，有顯著性的升高。IM患者當中，又以有感染幽門螺旋桿菌的IM患者有較高LOXL4甲基化。將LOXL4送入AGS細胞中，LOXL4的表達使AGS細胞的細胞群落形成分析 (colony formation assay) 的細胞群落減少，還有細胞增殖率也變慢。但是令人驚訝的是，經分析後發現，LOXL4的甲基化是一個獨立的預後指標，且LOXL4甲基化較高的病人其預後較好。與NR4A3不同，將LOXL4送入AGS細胞中，LOXL4的表達使AGS細胞的軟瓊脂分析 (soft agar assay) 的細胞群落增加。結論，我們的結果發現：胃癌中，異常JAK/STAT3訊息路徑導致NR4A3和LOXL4經由表基因而默化。且NR4A3和LOXL4的甲基化程度可以預測胃癌患者的存活率，並可能成為初步診斷胃癌的生物指標。

Gastric cancer is the second leading cause of cancer worldwide. Epigenetic silencing of tumor-suppressors has emerged as an important underlying mechanism in the gastric carcinogenesis. Previous studies showed that infection of H. pylori activates JAK/STAT3 signaling pathway in gastric cancer. However, the role of this aberrant signaling remains unclear. We hypothesized that activation of JAK/STAT signaling leads to epigenetic silencing of STAT3 target genes in gastric cancer. In our previous study, we has demonstrated that a constitutively active STAT3 led to epigenetic silencing of its downstream target genes, NR4A3 and LOXL4, via promoter CpG island hypermethylation in MKN28 cells where JAK/STAT signaling is intact. Ectopic expression of NR4A3 in AGS cells where JAK/STAT signaling is constitutively activated reduced cancer cell growth in colony formation assay (P<0.001), cancer cell growth in anchorage independent soft agar assay (P<0.05) and cell proliferation rate (P<0.001). In clinical specimens, quantitative methylated-specific PCR (qMSP) demonstrated a significant correlation between the degree of NR4A3 methylation and STAT3 nuclear translocation in gastric tumor samples (P<0.05). Importantly, methylation of NR4A3 was significantly associated with patients with shorter survival (P<0.05). In contrast, intestinal metaplasia (IM) patient demonstrated a significantly higher methylation of LOXL4 than in tumor and gastritis samples (P<0.001). IM patients infected with H. pylori have higher LOXL4 methylation than that without H. pylori (P<0.001). Ectopic expression of LOXL4 in AGS cells reduced cancer cell growth in colony formation assay (P<0.05) and cell proliferation rate (P<0.01). Surprisingly, multivariate COX-regression analysis showed that LOXL4 methylation is an independent prognostic marker and is associated with better prognosis in gastric cancer patients (HR=0.467; 95%(CI)=0.248-0.877; P=0.018). In contrast to NR4A3, ectopic expression of LOXL4 in AGS cells enhance cancer cell growth in anchorage independent soft agar assay (P<0.05), but did not affect the migration ability. In conclusion, our result demonstrated that aberrant JAK/STAT3 signaling induces epigenetic silencing of NR4A3 and LOXL4 in gastric cancer. Methylation of NR4A3 and LOXL4 predicts survival in gastric cancer patients and may be a biomarker for diagnosis of gastric cancer.

Contents
中文摘要 i
Abstract ii
Contents iv
List of figures vii
List of tables viii
Introduction 1
Gastric cancer 1
Helicobacter pylori (H. pylori) 1
The JAK/STAT signaling pathway 2
Epigenetic regulation 3
DNA methylation 3
Aberrant promoter hypermethylation and cancer 4
Aberrant JAK/STAT3 signaling and epigenetics 4
Nuclear receptor subfamily 4, group A, member 3 (NR4A3) 5
Lysyl oxidase-like 4 (LOXL4) 5
Objectives of study 7
Materials and methods 8
Cell cultures 8
Patient samples 8
Transient transfection 8
Stable transfection 9
Colony formation assay 9
Soft agar assay for anchorage independent cell growth 9
Cell viability assay for drug-resistance 10
Cell Proliferation assay 10
wound-healing assay for cell migration 10
RNA expression analysis 10
RNA extraction 10
Reverse transcription (RT) 11
Quantitative Real-time PCR (qRT-PCR) 12
DNA methylation analysis 12
DNA extraction 12
Bisulfite modification of DNA 13
Real-time quantitative methylation-specific PCR (qMSP) 14
Protein quantification analysis 14
Protein extraction 14
Protein quantification 15
Western blot 15
Results 17
Overexpression of LOXL4 inhibited colony forming activity of AGS cells 17
Promoter methylation of LOXL4 in clinical samples. 17
Higher LOXL4 promoter methylation status revealed a better patient overall survival outcome; no matter patients are in which stage. 18
Overexpression of NR4A3 and LOXL4 have no effect on the chemosensitivity of cisplatin in AGS cells 18
Overexpression of NR4A3 and LOXL4 in AGS cells affected the anchorage independent cell growth in soft agar assay. 19
Cell proliferation activity of NR4A3- and LOXL4-overexpressed stable clones was significantly decreased compared to vector-only control stable clone. 19
The cell migration ability of LOXL4-overexpressed stable clones cells did not increase compared to parental AGS cells. 19
Discussion 21
The roles of NR4A3 in gastric cancer 21
The roles of LOXL4 in gastric cancer 21
Methylation of NR4A3 and LOXL4 as biomarkers for gastric cancer prognosis 22
References 35

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