臺灣博碩士論文加值系統

胃癌是世界上胃癌死亡率的第二位，國人癌症死亡率的第六位，但其胃癌的致病機轉在醫學研究上尚未釐清，故我們一直在尋找新的有效的胃癌診斷分子來提高胃癌病人的存活率。近年來研究發現microRNA主要可以藉由其5’端的seed region和目標蛋白的3’未轉譯區域上的序列做配對，以達到抑制目標蛋白的轉錄或轉譯，進而負向調控下游目標基因的表現。目前許多證據不僅發現，microRNA在生物發育與組織分化中扮演非常重要的角色外，而且指出有些microRNA可以藉著所調控目標基因來達到它所扮演的onco-miR或tumor suppressor角色。首先，本實驗室利用microRNA qRT-PCR分析平台，從六對胃癌臨床檢體中建立270個microRNA profiling經由3-way-ANOVA統計分析後，進一步挑選Ct<35、p<0.05、表現量差異大於1.5倍以上，視為有統計意義的microRNA。挑選其中的miR-205為研究目標，miR-205在胃癌檢體中的表現量比在正常檢體中的表現量來的高2.41倍。進一步，大規模胃癌臨床檢體驗證中發現，其中有76.9%的臨床檢體其miR-205在胃癌檢體中的表現量比正常檢體來的高(n=52)，且miR-205的表現量與腫瘤淋巴結轉移(TNM)分期有統計上顯著意義，因此推測miR-205扮演onco-miR且促進轉移的角色。接下來，我們利用lentivirus expression system建立穩定過度體外表現miR-205或穩定抑制表現miR-205細胞株的穩定胃癌細胞株，藉由細胞移行實驗發現miR-205會促進胃癌細胞株的移行能力，同時利用之前實驗室建立之iTRAQ 定量蛋白體資料庫與線上軟體(miRBase)的資料庫進行交叉比對，找到GSTM1為miR-205的可能的目標基因，透過3端未轉錄區 luciferase reporter assay和西方墨點法 (Western blot)證實miR-205確實會去抑制其下游基因GSTM1的表現。此外，利用shRNA直接去抑制細胞內GSTM1的表現會促進為癌細胞株的細胞移行能力，證實GSTM1扮演tumor suppressor的角色。綜合以上結果證實miR-205是透過抑制GSTM1來促進細胞轉移，進而調控胃癌的致癌機轉。

Gastric cancer (GC) is the second leading cause of cancer-related death in the worldwide and the sixth in Taiwan . The tumorigenesis of GC is still unknown, and we are still search for the new biomarker to increase patients survival rate. Recent research indicate that microRNAs could negative regulate the target gene by binding to the 3’UTR of the target gene and inhibit the transcription or translation. microRNAs play important role in not only development and differentiation but tumorigenesis. Preview qRT-PCR profiling of 270 cancer related miRNAs in 6 pair gastric cancer clinical samples indicated that miR-205 was 2.41 time up-regulated in tumor with statistical significance(3-way-ANOVA , Ct<35, p<0.05, 1.5 fold change). In this study, miR-205 was selected as the candidate gene, which were up-regulated in 76.9% of clinical specimens in 52 pair samples, and statistical significantly with the TNM stage. We assumed that miR-205 may played as an onco-miR and correlated with tumor progression in GC. To investigate the biological function of miR-205, we used the lentiviral expression system to establish stable cell lines that were in vitro over-expressed or knocked down of miR-205. And the result indicated that miR-205 would promote cell migration in GC. To search for the potential target gene of miR-205, we correlated our preview iTRAQ database with online database (miRBase) and found Glutathione-s-transferase mu 1 (GSTM1). We validated that GSTM1 could negative regulated by miR-205 using 3’UTR luciferase reporter assay and western blot. In addition, in order to investigate whether GSTM1 could regulate cell migration in GC, we used shRNA to establish GSTM1 knocked down stable cell line and found out that GSTM1 could inhibit cell migration. The data suggest that GSTM1 played as a tumor suppressor. These results indicated that GSTM1 was one of the target gene that regulated by miR-205 to regulate the tumorigenesis of GC.

指導教授推薦書
論文口試委員審定書授權書
授權書 iii
誌謝 iv
中文摘要 v
ABSTRACT vii
目錄 ix
前言 - 1 -
胃癌 - 1 -
microRNA的生合成及生物功能 - 1 -
microRNA 205 (miR-205) - 2 -
miR-205在癌症所扮演的角色 - 2 -
miR-205的調控 - 3 -
Glutathione S-transferase mu 1(GSTM1) - 3 -
GSTM1與腫瘤 - 4 -
研究動機 - 6 -
材料與方法 - 7 -
I. 細胞株 (Cell Lines) - 7 -
II. 細胞Genomic DNA抽取 (Genomic DNA Extraction) - 7 -
III. 質體製作 - 7 -
IV. 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) - 10 -
V. 反轉錄病毒表現系統 (Lentiviral Expression System) - 10 -
VI. 細胞RNA抽取 (RNA Extraction) - 12 -
VII. 反轉錄聚合酶連鎖反應 (Reverse transcription Polymerase Chain Reaction ( RT-PCR) for microRNA) - 13 -
VIII. 即時定量聚合酶連鎖反應 (Real-time Polymerase Chain Reaction ( qPCR) for microRNA) - 13 -
IX. 3’端未轉錄區域luciferase report assay (3’UTR luciferase reporter assay) - 14 -
X. 細胞蛋白質抽取 (Protein Extraction) - 14 -
XI. 西方墨點法 (Western Blot) - 14 -
XII. 細胞移行實驗 (Migration Assay) - 16 -
XIII. 結果分析 (Satstic Analysis) - 16 -
結果 - 17 -
利用即時定量聚合酶連鎖反應確認胃癌病人臨床檢體組織中miR-205的表現量，並進行臨床病理上的統計分析 - 17 -
利用反轉錄病毒建立穩定過量表現miR-205或是穩定抑制miR-205表現的人類胃癌細胞株 - 18 -
miR-205對人類胃癌細胞株移行能力之影響 - 19 -
miR-205可能目標蛋白之預測 - 20 -
GSTM1於穩定過量表現miR-205或穩定抑制miR-205人類胃癌細胞株中的表現 - 20 -
利用反轉錄病毒建立穩定抑制GSTM1表現的人類胃癌細胞株 - 21 -
GSTM1對人類胃癌細胞株移行能力之影響 - 21 -
討論 - 23 -
參考文獻 - 28 -
實驗圖表 - 34 -
附錄 - 45 -

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