鑑定人類新微型核醣核酸及其調控基因

微型核醣核酸microRNA (miRNA) 是一群非常短的RNA，大約只有二十幾個鹼基，但卻具有影響基因表現的能力或蛋白質的生合成。miRNA與siRNA在生化機制及功能是相當類似的。很多證據指出miRNA能調控大多能轉錄出蛋白質的基因，因此，miRNA對於許多的細胞生理功能都扮演重大的調控，包括發育、細胞的增生與分化、細胞凋亡或癌症的生成與發展。近年來，約有五百多個miRNA基因被預測或透過實驗方式所驗證出來。由於目前推論出約大於一千多個miRNA基因存在人類基因組當中，而如何去發現或證實新的miRNA基因更是研究上的一大課題。李文雄院士以及施純傑博士的研究團隊發展透過組織作篩選的電腦分析的方式來預測新的miRNA基因及其調控的下游基因，因此我們能藉由實驗的方法在三種細胞株中作細胞驗證，分別是MCF-7乳癌細胞株、MCF-10A正常乳腺細胞、IMR-90肺部正常纖維母細胞，我們確認其中六種新的miRNAs存在於此三種細胞中，並進一步挑選其中兩種miRNA來作功能性及演化保留性分析，並利用冷光酶測定分析以及西方墨點法來驗證此新 miRNA的確存在並可以調控其下游基因的表現。

關鍵字

微型核醣核酸；調控基因；組織篩選；冷光酶活性分析

並列摘要

MicroRNAs are endogenous RNAs of 20-23 nucleotides in length that negatively regulate the expression of target genes by binding to the 3’-untranslated regions (UTRs) of the target mRNAs. The importance of miRNAs in animals is highlighted by the facts that they regulate a large proportion of protein-coding genes, and are involved in the control of a variety of processes including development, cell proliferation, tissue differentiation, apoptosis and metabolism. In addition, microRNAs are shown to be directly involved in cancer initiation and progression. Recent studies suggest that up to one third of human genes are regulated by miRNAs. However, although many human miRNA genes have been identified or predicted, it is believed that many more remain to be discovered. In this work, we identified the existence and measured the endogenous expression levels of novel miRNAs predicted by Dr. Wen-Hsiung Li and Dr. Arthur Chun-Chieh Shih; furthermore, we identified their target gene in different kinds of cell lines such as human breast cancer cell line MCF-7, non-tumorigenic MCF-10A and human lung fibroblasts IMR-90. In addition, we analyzed and verified the functions of the novel miRNAs using their mimics and inhibitors by luciferase reporter assay system and western blotting.

並列關鍵字

microRNA ； target gene ； tissue-selective ； luciferase assay

參考文獻

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2. Wightman, B., I. Ha, and G. Ruvkun, Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell, 1993. 75(5): p. 855-62.

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鑑定人類新微型核醣核酸及其調控基因

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