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Determining differentially expressed miRNAs and validating miRNA—target relationships using the SPRET/Ei mouse strain
- Timmermans, S., Van Hauwermeiren, F., Puimège, L., Dejager, L., Van Wonterghem, E., Vanhooren, V., Mestdagh, P., Libert, C., Vandenbroucke, R. E.
- Mammalian genome 2015 v.26 no.1-2 pp. 94-107
- Mus musculus, gene expression regulation, genes, inflammation, messenger RNA, mice, microRNA, microarray technology, neoplasms, phenotype, quantitative polymerase chain reaction
- Micro RNAs (miRs) are involved in many biological processes. The challenge of identifying genes influenced by miRs is evidenced by the relatively few validated miR–target interactions. In this work, we used the Mus spretus SPRET/Ei strain as an in vivo system to identify new miR–target relations. Mus spretus diverged from Mus musculus over one million years ago, making it genetically and phenotypically divergent. SPRET/Ei mice are resistant to inflammation and several cancers, making them attractive for different research fields. Their phenotype is unique and is considerably different from that of almost all other laboratory mouse strains. We exploited the characteristics of SPRET/Ei mice as a tool to identify miR–target relationships. Hepatic genes and miRs differentially expressed between C57BL/6 and SPRET/Ei mice at basal levels were identified with an Affymetrix microarray and a multiplex qPCR, respectively. A total of 955 genes and 38 miRs were identified as differentially expressed. Increased miR expression might result in downregulation of its target mRNA and vice versa. Subsequently, we used our miR and mRNA data to identify possible in vivo miR–target interactions. Ingenuity pathway analysis (IPA) analysis revealed 380 possible miR–target interactions. Five miRs were selected for experimental validation by in vivo overexpression of the miRs. This resulted in the confirmation of six previously unknown miR–target interactions: miR-146a, Zdhhc2; miR-150, Elovl3, Kcnk5, and Nrd1d2; miR-155, Camta1; and miR-592, Steap2. In conclusion, we show that SPRET/Ei mice can be used as a platform for miR–target identification in vivo, and we used this platform to identify and experimentally confirm miR–target interactions.