系統識別號 U0026-1407201015434900
論文名稱(中文) 利用TAG資料庫進行新穎癌症相關基因之研究
論文名稱(英文) Identification and characterization of novel tumor-associated genes in liver cancer
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 98
學期 2
出版年 99
研究生(中文) 洪偉翔
研究生(英文) Wei-Shiang Hung
學號 T1697405
學位類別 碩士
語文別 英文
論文頁數 49頁
口試委員 指導教授-孫孝芳
中文關鍵字 癌症相關基因  肝癌 
英文關鍵字 TAG  FRK  HCC 
中文摘要 先前我們實驗室於2006年時建立了一個Tumor-Associated Genes (TAG) 資料庫。這個資料庫到目前為止收錄了519個與癌症相關的基因,並且針對使用者提供相當完善的網路搜尋與分析的平台。此外,利用已經建立好的癌症相關蛋白質功能性區域之加權值量表去搜尋比對在人類資料庫中完整的cDNA序列,我們鑑定出一群具有癌化潛力的癌症相關基因。初步的分析顯示這些基因極有可能是新穎TAG。這個研究的目的是針對具有高度潛力的候選癌症相關基因進行進一步的研究來確認其在癌生成扮演的角色。藉由生物資訊學的工具以及分子生物學的實驗結果,我們把目標鎖定在Fyn-related kinase ( FRK ),一個在人類肝癌細胞株中高度表現的候選致癌基因上,並且去探討FRK在肝癌生成過程中所造成的癌化影響。透過使用68對臨床肝癌檢體進行西方墨點法(Western blotting)分析,結果呈現出高達52% (35/68)比例的肝癌檢體可偵測到FRK有高度表現的情形。比較其臨床病理上的特徵表現,發現到FRK的表現量和病毒感染有關,特別是B型肝炎病毒。進一步我們使用兩株不同的肝癌細胞株,Hep3B與HepG2,讓FRK在這兩株細胞株內大量表現來研究其癌化的能力。利用不同的癌症分析方法,包括細胞增生、細胞侵襲與細胞轉型等分析,我們觀察到在FRK大量表現下可以促使Hep3B細胞(具有B型肝炎病毒)成為具有高度增生、侵犯與轉型能力的細胞。然而FRK大量表現在不具有B型肝炎病毒的HepG2細胞中卻只有提高侵犯能力。總結這些研究結果,我們認為FRK為一個有潛力的新穎致癌基因,如大量表現在B型肝炎病毒感染的肝細胞中可能會加速肝癌的形成。由這個研究的結果我們也驗證了TAG資料庫預測新穎癌症基因的準確性。相信TAG資料庫可提供未來在鑑定其他的新穎基因時的一個好的研究工具。
英文摘要 Previously our laboratory has established a tumor-associated genes (TAGs) database to provide user-friendly environment for searching and analyzing TAG information online. By using the TAG domain signature profile, we screened the expressed sequences in the whole human genome and identified a group of genes that are potentially involved in tumorigenesis. This study aimed to characterize the selected novel TAG, Fyn-related kinase (FRK), and study its role in tumorigenesis. Preliminary test showed FRK mRNA was over-expressed in liver cancer and Western blot confirmed the FRK protein expression in liver cancer cell lines. Thus we focused on to study the tumorigenic effect of FRK in hepatocellular carcinoma (HCC). Using 68 paired HCCs, our data demonstrated that FRK is up-regulated in 52% (35/68) of cases. The clinicpathological comparison showed the expression level of FRK was significantly associated with HBV infection. Further functional characterization was carried out in two HCC cell lines, Hep3B and HepG2, overexpressing FRK. Results from various tumorigenic assays showed that FRK-overexpression promotes cell growth, invasiveness and transformation abilities in HBV-positive Hep3B cells. Nevertheless, overexpressed FRK only increase cell invasion in HBV-negative HepG2 cell. Taken together, these findings suggested that FRK promotes liver cancer formation and the effect is enhanced by the presence of HBV. Furthermore, our data also demonstrated the accuracy of TAG prediction and suggested the other predicted candidate TAGs can be potential target for future cancer research.
論文目次 中文摘要 I
Abstract II
誌謝 III
Table of contents V
List of tables VIII
List of figures IX
1. Introduction 1
1.1 Introduction of cancer and causing of cancer 1
1.1.1 Chemical and physical mutation promote tumorigenesis 2
1.1.2 Virus infection induce cancer formation 2
1.1.3 Heredity mutation and cancer 3
1.1.4 The relationship between cancer and associated genes 3
1.2 The Tumor-associated gene (TAG) Database 4
1.3 Identification of potential TAGs 5
1.3.1 Preliminary study of potential TAGs. 5
1.3.2 Digital confirmation of predicted TAGs 5
1.3.3 Experimental confirmation of potential TAGs 6
1.4 Objective of this study 6
2. Materials and Methods 7
2.1 Database update 7
2.2 Selection of candidate tumor associated genes 7
2.3 Total RNA extraction 7
2.4 Reverse transcription 8
2.5 Polymerase chain reaction (PCR). 8
2.6 Protein extraction and concentration determination 8
2.7 Antibodies 9
2.8 Western blotting 9
2.9 cDNA panel 10
2.10 Clinical samples 10
2.11 Cell lines 10
2.12 Plasmid DNA preparation 11
2.13 Plasmid DNA transfection 12
2.14 Immunocytochemistry 12
2.15 Cell Proliferation assay 13
2.16 Cell Invasion assay 13
2.17 Colony Formation assay (Soft agar assay) 14
2.18 Statistical analysis 14
3. Results 16
3.1 TAG database update 16
3.2 Select the candidate TAGs for further study 16
3.2.1 Select the candidate genes via bioinformatics tools 17
3.2.2 Select the candidate genes via molecular biology experiment 17
3.3 Examine the expression of FRK in cDNA and protein in HCC cell lines 18
3.4 FRK is overexpressed in paired HCC samples 18
3.5 Functional analysis of FRK effect 19
3.5.1 Transient transfection of overexpressing FRK construct 19
3.5.2 The cell proliferation analysis under over-expression FRK 19
3.5.3 The cell invasion analysis under over-expression FRK 19
3.5.4 The cell transformation analysis under over-expression FRK 20
4. Discussions 21
4.1 Characterization of FRK, a non-receptor tyrosine kinase 21
4.1.1 The introduction of protein tyrosine kinase 21
4.1.2 The identification of Fyn related kinase (FRK) 22
4.1.3 The distribution and structural feature of FRK 22
4.2 FRK and cancers 23
4.2.1 FRK and tumor suppressor pRb 23
4.2.2 FRK associated with tumor suppressor PTEN 24
4.2.3 FRK and ETV6 24
4.2.4 FRK may play dual roles in tumorigenesis 25
4.3 The relationship between Hepatitis B virus infection and FRK 26
4.4 Tumor-associated genes database, a useful tool for searching novel TAGs 28
5. Conclusion 29
6. Reference 45
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