Functional screening using a microRNA virus library and microarrays: a new high-throughput assay to identify tumor-suppressive microRNAs
Functional screening using a microRNA virus library and microarrays: a new high-throughput assay to identify tumor-suppressive microRNAs
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DOI：
10.1093/carcin/bgq112
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发表时间：
2010-08-01
期刊：
影响因子：
4.7
通讯作者：
Nakagama, Hitoshi
中科院分区：
文献类型：
--
作者：
Izumiya, Masashi;Okamoto, Koji;Nakagama, Hitoshi
MicroRNA（miRNA）是一类转录后抑制靶信使RNA表达的非编码RNA。越来越多的证据支持它们在各种正常细胞过程以及病理条件（如癌症）中的作用。我们建立了一种功能性筛选试验，通过组合表达数百种miRNA前体的混合慢病毒载体和定制的微阵列，能够高通量鉴定在感兴趣的癌症表型中起作用的miRNA。使用合并的聚合酶链反应产物进行自身与自身杂交分析，产生高度线性和可重复的结果。为了测试该分析的可行性，我们专注于控制胰腺癌细胞增殖的miRNA，并成功鉴定了五种负控制细胞增殖的miRNA，包括先前被鉴定为代表性肿瘤抑制miRNA的miRNA-34 a。使用表达五种miRNA或合成miRNA中的每一种的慢病毒载体进一步验证结果。该测定的基于功能的性质使得能够鉴定与细胞增殖密切相关的miRNA，但是该测定的相对容易性和灵活性允许将来对癌症干细胞、转移和其他感兴趣的癌症表型进行研究。
MicroRNA (miRNA) is a class of non-coding RNAs that represses expression of target messenger RNAs posttranscriptionally. A growing body of evidence supports their roles in various normal cellular processes, as well as in pathological conditions, such as cancer. We established a functional screening assay that enables high-throughput identification of miRNAs that have a role in cancer phenotypes of interest, via the combination of pooled lentivirus vectors expressing several hundred miRNA precursors and a custom-made microarray. Self versus self-hybridization analysis using pooled polymerase chain reaction products generated highly linear and reproducible results. To test the feasibility of the assay, we focused on miRNAs that control proliferation of pancreatic cancer cells and successfully identified five miRNAs that negatively control cell proliferation, including miRNA-34a that was previously identified as a representative tumor-suppressive miRNA. The results were further validated using lentivirus vectors expressing each of the five miRNAs or synthetic miRNAs. The function-based nature of the assay enabled identification of miRNAs that were strongly linked to cell proliferation, but the relative ease and flexibility of the assay allow for future studies of cancer stem cells, metastasis and other cancer phenotypes of interest.