title: A simple high-throughput technology enables gain-of-function screening of human microRNAs.
abstract: 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.
authors: Center for Stem Cell Biology & Regenerative Medicine, University of Maryland School of Medicine, Baltimore, MD, USA.
authors: Wen-Chih
authors: WC
authors: Cheng
authors: Tami J
authors: TJ
authors: Kingsbury
authors: Sarah J
authors: SJ
authors: Wheelan
authors: Curt I
authors: CI
authors: Civin
mesh_terms: D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D059467:Transcriptome
chemical_list: D035683:MicroRNAs
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