- 學位論文
利用雙色螢光報導子線蟲探討let-7/lin-41路徑中的可能的調控因子
Using a dual-fluorescence 3′ UTR reporter to investigate putative regulators in the C. elegans let-7/lin-41 pathway
摘要
秀麗隱桿線蟲（Caenorhabditis elegans）的生長與發育主要由一連串具有時間性的基因所控制，這群基因被稱為異時性基因（Heterochronic genes），其中包括特定的微小核糖核酸（micro RNAs）以及RNA結合蛋白。其中，具有高度保留性的微小核糖核酸let-7便是影響到線蟲由幼蟲進入到成蟲階段的一個重要因子，let-7的缺失會使線蟲在成蟲時出現接縫細胞（epithelial stem-like seam cell）重複分裂、生殖孔爆裂等性狀。在人類中，let-7的缺失同樣會影響細胞的分化，導致癌症的發生。let-7在線蟲中主要由LIN-28所調控，並會抑制下游lin-41的表現。而lin-41的降低會使LIN-29開始表現，因此let-7的缺失會間接導致LIN-29的表現被抑制。 Musashi蛋白是一種RNA結合蛋白，主要被發現於幹細胞中，含量過高時與癌症的發生有很高的相關性。先前有研究指出，在小鼠胚胎幹細胞中，MSI-1可能會透過結合LIN-28蛋白間接調控下游let-7 miRNA的生成，讓我們對於MSI-1在線蟲中與let-7的關聯性與異時性途徑產生興趣。為了探討MSI-1對於異時性途徑的影響，我們利用會同時表現兩種由不同3′ UTR調控的雙色螢光報告子的線蟲，GFP由lin-41 3′ UTR所控制，而mCherry由不受let-7影響的unc54 3′ UTR所調控，藉由螢光的變化去觀察利用小核糖核酸干擾技術去降低MSI-1表現時是否對let-7功能造成影響。可惜的是，就我們初步的觀察，MSI-1的下降並不會對於蟲的let-7對lin-41 3′ UTR reporter 的調控表現量以及其變異的性狀造成影響。 與此同時，我們也利用雙色螢光報告子線蟲在具有潛力的基因中，利用篩選新的研究目標。透過雙色螢光蟲的GFP變化，我們發現有兩個基因ani-1和mix-1表現量可能會影響到let-7對於lin-41 3′ UTR的控制，但在利用北方墨點法做檢查以後，發現它們與let-7表現量沒有關係。但對於它們是否透過其他的途徑去影響到seam cells的變化，仍有待未來進一步探討。
並列摘要
In Caenorhabditis elegans, the development is controlled by a series of genes that show up at specific moments, including microRNAs and RNA binding proteins. These genes are called “heterochronic genes”. Among the genes, a highly conserved micro RNA let-7 plays the key role in the switch of worms from juvenile to adult. let-7 shows up at late L3 stage of C. elegans and promotes the differentiation of tissue by inhibiting downstream genes like lin-41. The defect in let-7 would cause several retardations in the worms, the notable phenotype includes the repetitive division of epithelial stem-like seam cells and vulva bursting. The temporal regulation of let-7 in development is conserved in invertebrates and vertebrates. In addition, reduced let-7 expression leads to the overexpression of many oncogenes and eventually promotes the progression of cancer. Musashi protein is a conserved RNA-binding protein which is first discovered in neuronal stem cells of Drosophila. In human, overexpression of musashi protein has been implicated to cancers in the gut, central nerve system and hematopoietic cells. As for C. elegans, MSI-1 is also expressed in nervous system and has been reported to control memory loss and the mating behavior. Previously, in mouse embryonic cells, MSI-1 has been reported to interact with LIN-28 and may promote inhibition of let-7 biogenesis, which inspires us to explore the relationship of MSI-1 and let-7 in heterochronic pathway of C. elegans. To study this, here we employ a dual fluorescence 3′ UTR reporter in C. elegans with the 3′ UTR of lin-41, a major target of let-7, fused to GFP while the unc-54 3′ UTR fused to mCherry as a control. We observed and measured changes in GFP signal upon msi-1 knockdown but has not found significant changes. At the same time, we used the dual-fluorescence worms to screen a pack of potential regulators of let-7 biogenesis of function that were revealed by previous RNAi screening studies. By observing the GFP pattern of the dual-fluorescence worms, we found two genes, ani-1 and mix-1, whose expression were likely to affect let-7 controlling lin-41 3′ UTR. The results from northern blot assays indicated that the levels of let-7 miRNA are not changed. Thus, these two genes may not regulate let-7 biogenesis but could affect other factors required for the let-7 miRNA to function properly. Further investigation for the roles of ani-1 and mix-1 in let-7 repressing lin-41 awaits.