2013-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643314摘要：微小核醣核酸 (mircoRNAs) 是微小單股具調節性的核醣核酸，控制細胞分裂，分化及凋亡等發育過程。在線蟲 C. elegans 中，let-7 直接調節癌症基因 let-60/RAS，亦調節 lin-41，其為人類PML的同源基因，在人類中PML的突變可以在各型前骨髓性細胞白血病中發現。在線蟲let-7突變種中，表皮接縫細胞 (epidermal seam cells) 在進入成蟲階段時無法脫離細胞週期而重複其在幼蟲階段的細胞分裂，而缺乏對細胞週期的控制與無法正確細胞分化正是癌症的特點之一。人類 let-7 調節癌症基因 RAS，且人類 let-7 的表現缺失已證實與肺癌與乳癌有關連。在本研究計畫中，我們將鑑定調控 let-7作用的因子，其本身極可能也與癌症相關。ALG-1 是線蟲微小核醣核酸基因沉默複合體中的重要作用蛋白，可直接與包括let-7在內的微小核醣核酸結合，其本身對於let-7發揮功能是需要的。我們將利用免疫沈澱法與蛋白質譜儀分析來鑑定ALG-1之共同作用蛋白，並進一步分析他們可能在let-7功能中所扮演的角色。我們的初步結果顯示Receptor of Activated C Kinase (RACK-1) 與ALG-1 蛋白結合並且在let-7功能中扮演負向調控的角色。另一方面，我們先前研究結果顯示，let-7可以與特異性蛋白結合並形成具特異性的核酸蛋白複合體，因此我們將使用反序核醣核酸將內生的let-7結合純化並鑑定分析與其結合的蛋白分子。除了以上利用蛋白體學方法的研究外，我們將使用正向遺傳學篩選，利用篩選合成致死或抑制let-7突變的基因來尋找新奇的let-7功能調控因子。由於 let-7 在人類中具遺傳保守性並且與癌症有直接相關，這項計畫的研究成果可以幫助了解 let-7 在癌症生成過程中所扮演的角色，亦有助於未來在癌症治療上的應用。<br> Abstract: MicroRNAs (miRNAs) are small single-stranded regulatory RNAs that control essential development processes such as cellular differentiation, proliferation, and apoptosis. In C. elegans, let-7 directly regulates let-60/RAS, a common human oncogene, and another gene, lin-41, which is homologous to mammalian cancer genes, including PML, mutated in almost all cases of promylocytic leukemia. In let-7 mutant, epidermal seam cells frequently fail to terminally differentiate and instead elect to divide again, which is a hallmark of cancer. In humans, the let-7 miRNA regulates the RAS oncogene and defective expression of let-7 is associated with lung and breast cancers. In this proposal, we search for novel functional modulators of let-7, which are themselves likely to be cancer-related. The C. elegans Argonaute protein, ALG-1, is the key player in the miRNA-induced silencing complex (miRISC). ALG-1 associates with miRNAs, including let-7, and is required for proper let-7 function. We will identify ALG-1 co-factors by immunoprecipitation and mass spectrometry and further determine their possible roles in let-7 function. Our preliminary results have shown that Receptor of Activated C Kinase (RACK-1) associates with ALG-1 and plays a negatively regulatory role in let-7 function. On the other hand, since our previous results indicate that let-7 assembles with specific ribonucleoprotein complexes and may have its own set of specific binding proteins, we will directly isolate endogenous let-7-binding complexes by anti-sense oligonucleotide pull-down assay and analyze the associating proteins. Besides the proteomic approaches, we will also employ forward genetics screens to search for novel functional modulators of let-7 based on the principle of mutation suppression or synthetic lethality.Since let-7 is conserved in human and is associated with cancers, our accomplishment of this project will shed light on the roles of miRNAs in carcinogenesis and lead to the future applications in cancer therapy.微小核醣核酸線蟲microRNAC. elegans