胃癌在2012 世界衛生組織的統計中發生率為第五名，其中以胃腺癌為主。在2014
年美國癌症基因體圖譜計畫(TCGA)中針對胃癌進行多種分子鑑定將胃癌區分成四種
子型(人類泡疹病毒第四型關連、微衛星不穩定、染色體不穩定以及基因體穩定)。
在本研究中，我們與長庚醫院合作，針對19 個胃癌(包括6 個腸道型胃癌、7 個瀰
漫型胃癌、6 個混合型)檢體進行409 個癌症相關基因次世代定序。與TCGA 的結果
相同的發現在RTK/RAS/PI(3)K、TGF-β、β-catenin 訊息傳遞路徑以及細胞週期有高度的基因變異，其中調控細胞增生和爬行的RTK/RAS/PI(3)K 訊息傳遞路徑變異最大。在第二批76 個胃癌檢體(包括35 個腸道型胃癌、28 個瀰漫型胃癌、12 個混合型、1 個未知)中鎖定Kras 做進一步的次世代定序分析。整合95 個胃癌檢體中，共發現四個基因變異，包括G12A (1.05%)、G12D (1.05%)以及G13D (2.11%)。在與臨床病理資料分析發現Kras 基因變異較常出現於賁門區域(p = 0.022)，且較常為腸道型胃癌。在細胞實驗中，我們發現Kras G12A、G12V 和G13D 基因變異會導致細胞型態呈現紡錘狀且增強其爬行能力。總結，本篇研究發現台灣胃癌病患有4.04% Kras基因變異，屬於少數，其中變異位置皆位於 P-loop 上，此類腫瘤較常出現於賁門附近，在細胞實驗中發現這些變異會增家癌細胞的爬行能力。

Gastric cancer (GC) is the fifth most common cancer in 2012 and most of the case is adenocarcinomas. The Cancer Genome Atlas (TCGA) project of GCs has defined four subtypes of mutational signatures (Epstein–Barr virus-associated, microsatellite instability, chromosomal instability, and genomically stable signatures).We first performed ultra-deep targeted sequencing analysis of 19 gastric tumors (intestinal, n = 6; diffuse, n = 7; mixed, n = 6) to screen for mutations in 409 cancer-related genes in collaboration with CGMH. TCGA and our data revealed that the high alteration on three pathways, RTK/RAS/PI(3)K, TGF-β, β-catenin pathway and cell cycle. RTK/RAS/PI(3)K which controls cell
proliferation and migration had the highest alterations. We further characterized Kras mutation for 76 GC cases (intestinal, n = 35; diffuse, n = 28; mixed, n = 12; unclassified, n = 1). Of 95, mutation was detected in four cases (4.21%): G12A (1.05%), G12D (1.05%), and G13D (2.11%). Analysis of clinicopathological parameters revealed that Kras mutation gastric cancer appeared in cardia gastric more with p = 0.022. Intestinal type GC has higher
Kras mutation rate than diffuse type. Functional characterization of Kras mutations G12A, G12V and G13D in MKN28 revealed a higher level of migratory activity and a spindle-like phenotype than wild type. Together, this study reports a small proportion (4.21% = 4
of 95) of GC patients who carry specific Kras P-loop mutations associated with cardia and elevated migration activities.

Index
中文摘要 ii
Abstract iv
List of abbreviation vi
List of tables 4
List of figures 5
1 Introduction 7
1.1 Gastric cancer 7
1.2 Gastric cancer progression 8
1.3 Genetic alterations of gastric cancer 9
1.4 Ras family 10
1.5 Kras 10
1.6 Current treatment for Kras mutation 12
1.7 Purpose of this research 14
2 Materials and methods 15
2.1 Cell culture and cell lines 15
2.2 Western blot analysis 15
2.3 RNA extraction 16
2.4 Real-time quantitative PCR 17
2.5 Genotype sequencing 18
2.6 Lentivirus production 19
2.7 Lentiviral shRNA experiments 19
2.8 Ras Dependency Index (RDI) 20
2.9 Construction of Kras mutations stable cell lines 21
2.10 Cell proliferation assay 21
2.11 Clonogenic assay 22
2.12 Wound healing assay 22
2.13 Transwell migration assay 22
2.14 Statistical analysis 23
3 Results 24
3.1 Sample collection 24
3.2 Somatic genome alterations 24
3.3 Kras gene alteration in GC 26
3.4 Kras expression and mutation in gastric cancer cell lines 27
3.5 Determined the Kras dependency of gastric cancer cell lines 27
3.6 Correlation between Kras dependency and protein expression level 29
3.7 Kras G12A, G12V, G13D didn’t affect proliferation in MKN45/MKN28 gastric cancer 29
3.8 Kras G12A, G12V, and G13D MKN28 induced morphological change and had an increased level of migration. 31
4 Discussion and Conclusion 32
5 References 37

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