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Determining differentially expressed miRNAs and validating miRNA--target relationships using the SPRET/Ei mouse
S Timmermans1, F Van Hauwermeiren, L Puimège
1Inflammation Research Center (IRC), VIB-Ghent University, Technologiepark 927, Zwijnaarde, 9052, Ghent, Belgium, steven.timmermans@irc.VIB-UGent.be.
Abstract:
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.
Insights
This study introduces Mus spretus mice as a novel in vivo system for discovering micro RNA (miR)-target interactions. Researchers identified and validated six new miR-target relationships, advancing our understanding of gene regulation.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- Micro RNAs (miRs) regulate numerous biological processes, but identifying their target genes remains challenging, with few validated interactions.
- The Mus spretus SPRET/Ei mouse strain, genetically divergent from Mus musculus, exhibits unique resistance to inflammation and cancer, making it a valuable model for biological research.
Purpose of the Study:
- To utilize the unique genetic and phenotypic characteristics of Mus spretus SPRET/Ei mice as an in vivo system for identifying novel micro RNA (miR)-target interactions.
- To experimentally validate predicted miR-target relationships discovered using this model.
Main Methods:
- Differential gene expression analysis between C57BL/6 and SPRET/Ei mice using Affymetrix microarrays for hepatic genes.
- Differential micro RNA (miR) expression profiling using multiplex quantitative PCR (qPCR).
- Bioinformatic analysis (Ingenuity Pathway Analysis - IPA) of combined miR and mRNA data to predict interactions, followed by experimental validation of selected miRs via in vivo overexpression.
Main Results:
- Identification of 955 differentially expressed genes and 38 differentially expressed miRs between the two mouse strains.
- Prediction of 380 potential miR-target interactions using bioinformatic analysis.
- Experimental confirmation of six novel miR-target interactions involving miR-146a, miR-150, miR-155, and miR-592.
Conclusions:
- Mus spretus SPRET/Ei mice serve as an effective platform for in vivo micro RNA (miR)-target identification.
- The study successfully identified and experimentally validated several previously unknown miR-target interactions, contributing new knowledge to gene regulation mechanisms.
