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系統識別號 U0026-2108201409463300
論文名稱(中文) Fibronectin聚集相關之微小RNA miR-146a經由抑制vimentin蛋白抑制食道癌細胞爬行及轉移
論文名稱(英文) Fibronectin assembly-related miR-146a suppresses motility and metastasis through targeting vimentin in esophageal squamous cell carcinoma
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 102
學期 2
出版年 103
研究生(中文) 李季樺
研究生(英文) Chi-Hua Lee
學號 s46014116
學位類別 碩士
語文別 英文
論文頁數 53頁
口試委員 指導教授-劉校生
口試委員-王憶卿
口試委員-鄭宏祺
口試委員-吳明蒼
中文關鍵字 纖連蛋白  細胞爬行  食道癌  微小RNA  波形纖維蛋白 
英文關鍵字 cell migration  ESCC  miR-146a  metastasis  vimentin 
學科別分類
中文摘要 食道癌2013年於世界排名第五而台灣排名第九之癌症,依組織型態區分為鱗狀上皮細胞食道癌(ESCC)及腺癌(EA),在台灣95%為ESCC。然而食道癌難以治癒的最大原因為其易轉移的腫瘤細胞及早期診斷上困難,導致五年存活率低(小於15%)。但目前對食道癌轉移的機制並不清楚,因此本研究主要為探討食道癌中微小核糖核酸調控癌細胞轉移的機制。微小核糖核酸為小型非編碼的核糖核酸,會結合到目標基因的3'非編碼區去降解訊息核糖核酸或是去抑制轉錄作用。微小核糖核酸能為抑制或誘發腫瘤角色,也是轉移作用的調控者。先前研究中我們發現纖連蛋白(fibronectin),一個具多功能黏附型醣蛋白,能聚集在細胞膜表面,參與癌發過程中如細胞爬行和入侵作用。我們先前已發現大量的纖連蛋白與miR-146a具負相關。本研究中發現miR-146a會專一性結合它的目標基因-波形纖維蛋白(vimentin)。波形纖維蛋白為間葉組織細胞的標記並參與epithelial-to-mesenchymal transition (EMT)現象。我們更進一步利用食道癌細胞CE81FN+證實miR-146a會透過抑制vimentin表現抑制細胞爬行及入侵能力。從臨床食道癌病人檢體上也證實miR-146a及vimentin皆具顯著重要性。藉由IHC和ISH染色檢測檢體中兩者蛋白質表現量,結果顯示較高表現vimentin的腫瘤組織中,miR-146a的表現較低。並發現vimentin與miR-146a皆與病人兩年存活率相關。在小鼠實驗的部分,先前研究發現此食道癌細胞株於小鼠體內形成腫瘤能力長至數月久,為此我們建立分別帶有冷光及IPTG-inducible K-ras致癌基因的食道癌細胞株。前者能應用於活體實驗的腫瘤觀察,而後者之目的在於增加癌細胞形成腫瘤能力,未來會以此做更進一步小鼠實驗。另外將miR-146a過度表現的細胞株送microarray之分析出一群上升級下降基因,未來並將會以此再結合connectivity-map線上資料庫來預測具有抑制食道癌轉移潛力的藥物。綜述之,與纖連蛋白呈負相關係的miR-146a能為腫瘤抑制角色並且透過抑制vimentin影響食道癌細胞爬行及入侵的作用。
英文摘要 MicroRNAs (miRNAs) are small non-coding RNAs, which bind to 3’UTR of the target gene to degrade mRNA or inhibit translation. MiRNA acts as either a tumor suppressor or an oncogene. Esophageal cancer is the fifth leading cause of cancer death worldwide and ranks the ninth in Taiwan in 2013. There are two major histological types of esophageal cancers: Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EA). Over 95% of the esophageal cancers in Taiwan are ESCC. The major risk factor of ESCC is the metastatic spread of tumor cells, which results in poor prognosis and low 5-year survival rate (less than 15%). However, the underlying mechanism of metastasis in ESCC remains unclear. Fibronectin (FN), a multifunctional adhesive glycoprotein which assembles on the cell membrane, contributes to carcinogenesis including cell migration and invasion. We previously showed that the abundance of FN assembly on ESCC cell membrane is negatively correlated with miR-146a expression. In this study, we identified that vimentin, which is a mesenchymal marker and involves in epithelial mesenchymal transition (EMT), is the target gene of miR-146a. We further proved that miR-146a suppresses both migration and invasion of CE81FN+ cell through inhibiting vimentin by Real-Time PCR and transwell assays, respectively. The importance of miR-146a and its target gene vimentin in clinical specimens was demonstrated by IHC staining followed by statistical analysis. MiR-146a mediated suppression of metastasis was also detected in another ESCC cell line (KYSE). In conclusion, we reveal that FN assembly-negatively related miR-146a acts as a tumor suppressor to inhibit ESCC cell migration and invasion by targeting vimentin.
論文目次 Contents
Chinese Abstract…………………………………………………………………I
Abstract……………………………………………………………………………II
Acknowledgements…………………………………………………………III
Contents……………………………………………………………………………IV
Table and Figure List………………………………………………………VI
Abbreviation List…………………………………………………………VIII
Introduction
I. Esophageal Squamous Cell Carcinoma (ESCC)……………………1
II. The Relationship between Fibronectin (FN) and ESCC… 1
III. MicroRNA …………………………………………………2
IV. The Relationship between MicroRNA and ESCC…………………3
V. The Relationship between K-ras oncogene and ESCC…………3
VI. Specific aims………………………………………………………………3
Materials and Methods
I. Cell lines culture………..………………………………………… 5
II. Tissue specimens…..………………………………………………………5
III. Establishment of transient and stable cell lines………5
IV. Western blot analysis………..…………………………………6
V. Detection of miRNA and mRNA by real-time polymerase chain reaction (PCR)..…………………………………………………… 7
VI. Transwell assay for cell migration and invasion assays …………8
VII. Immunofluorescence staining……………………………..………8
VIII. Inducible plasmid K-ras construction………………………9
IX. Immunohistochemistry staining (IHC) staining……..……9
X. MiRNA in situ hybridization (miRNA ISH)…………..…………10
XI. For in vivo study……………..………………………………………11
XII. Statistical analysis………………………...……………… 11
Results
I. High fibronectin assembly was correlated with cell migration and miR–146a is most down-regulated in fibronectin abundantly assembled CE81T cells…….... 12
II. Low miR-146a expression is correlated with high CE81-FN+ cell migration...... 12
III. Vimentin is the target gene of MiR-146a………………… 13
IV. Vimentin expression was positively correlated with fibronectin assembly but negatively correlated with miR-146a expression……………………..………. 14
V. MiR-146a inhibits ESCC cell migration and iinvasion ithrough itargeting vimentin……………………………………… 14
VI. MiR–146a and vimentin expression levels correlate with ESCC tumor stage and survival rate of ESCC patients.…………………15
VII. MiR-146a suppresses metastasis in a xenograft mouse model.……………...... 16
VIII. CE81FN+k-ras cell lines stably expressing an inducible K-ras oncogene was established…………………………17
Discussion………………………………………………………… 19
Conclusion………………………………………………………………………… 24
References……………………………………………………………………… 25
Tables……………………………………………………………………………… 31
Figures……………………………………………………………………………… 38




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