2013-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659202摘要：微小RNA近來被認為在許多生物作用扮演重要角色，包括腫瘤的發生。癌症 細胞的微小RNA出現全面性的減少，顯示大多數的微小RNA會抑制癌症。 最近Lin-41被發現是微小RNA蛋白質Ago2的E3 ligase。我們的初步分析發 現Lin-41在肝細胞癌過度表現。因此，我們假設Lin-41藉由對經由微小RNA 的基因靜默的去抑制而促進腫瘤生成。為了證明此假說，我們將進行以下實 驗：(1)以即時反轉錄-聚合酵素鏈反應分析250對肝細胞癌的Lin-41表現，以研 究其臨床病理意義，和它是否可以當預測病人預後的生物標記。(2)在人類肝細胞癌細胞株過度和減少Lin-41的表現，以MTT,軟膠和免疫 不全小鼠注射試驗去研究Lin-41表現對腫瘤生長的影響。(3)使用螢光酵素測定去證明經由微小RNA的基因靜默會被Lin-41抑制。再 以西方墨潰法，共沉澱，和細胞内及細胞外泛素化試驗證明Ago-1和Ago-2 兩者都是Lin-41的受質。(4)研究Ago-1和Ago-2對腫瘤生長的角色。(5)以西方墨潰法和微陣列法找出Lin-41活化的致癌途徑。<br> Abstract: MicroRNA (MiRNA) is increasing recognized to play important roles in many biological processes, including neoplastic transformation. Global reduction of miRNA is found in cancers, indicating majority of miRNA are tumor suppressors. Lin-41 is recently found to be an E3 ligase for microRNA protein Ago2. Our preliminary data showed Lin-41 is overexpressed in hepatocellular carcinoma (HCC). Therefore, we hypothesize Lin-41 enhances neoplastic transformation by derepression of miRNA-mediated gene silencing. To prove this hypothesis, we plan to perform the following experiments:(1) Analyze the expression of Lin-41 in 250 HCC by real time RT-PCR to study the clinicopathologic significance of Lin-41 expression and its role as a biomarker for predicting patient prognosis.(2) Overexpression and knockdown of Lin-41 in human HCC cell lines and study its effect on tumor growth by MTT, soft agar, and SCID mice injection assays.(3) Use luciferase assay to prove miRNA mediated gene silencing is inhibited by Lin-41 and prove both Ago1 and Ago2 are substrates of Lin-41 by western blot, co-immunoprecipitation, in vivo and in vitro ubiquitination assay.(4) Study the role of Ago-1 and Ago-2 in tumor growth.(5) Identity the oncogenic pathway activated by Lin-41 by western blot and microarray.Lin-41微小RNARISC 複合體腫瘤生長Lin-41microRNARISC complextumor growth