科研之友
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摘要
MicroRNAs (miRs) regulate cellular processes by modulating gene expression. Although transcriptomic studies have identified numerous miRs differentially expressed in diseased versus normal cells, expression analysis alone cannot distinguish miRs driving a disease phenotype from those merely associated with the disease. To address this limitation, we developed miR-HTS, a method for unbiased high-throughput screening of the miRNome to identify functionally relevant miRs. Herein, we applied miR-HTS to simultaneously analyze the effects of 578 lentivirally transduced human miRs or miR clusters on growth of the IMR90 human lung fibroblast cell line. Growth-regulatory miRs were identified by quantitating the representation (i.e., relative abundance) of cells overexpressing each miR over a one-month culture of IMR90, using a panel of custom-designed quantitative real-time PCR (qPCR) assays specific for each transduced miR expression cassette. The miR-HTS identified 4 miRs previously reported to inhibit the growth of human lung derived cell lines and 55 novel growth-inhibitory miR candidates. Nine of 12 (75%) selected candidate miRs were validated and shown to inhibit IMR90 cell growth. Thus, this novel lentiviral library-and qPCR-based miR-HTS technology provides a sensitive platform for functional screening that is straightforward and relatively inexpensive.
- 出版日期2013-2
