Functional screening using a microRNA virus library and microarrays: a new high-throughput assay to identify tumor-suppressive microRNAs.
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.
Izumiya
Masashi
M
National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.
Okamoto
Koji
K
Tsuchiya
Naoto
N
Nakagama
Hitoshi
H
D002453:Cell Cycle; D002455:Cell Division; D045744:Cell Line, Tumor; D017931:DNA Primers; D005434:Flow Cytometry; D015723:Gene Library; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000473:pathology; D010641:Phenotype; D016133:Polymerase Chain Reaction; D013234:Stem Cells / Q000473:pathology; D013601:T-Lymphocytes
D017931:DNA Primers; D035683:MicroRNAs