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Experimental identification of miR-378-3p targets by overexpression and silencing in murine NIH-3T3 fibroblasts

ABSTRACT: MicroRNAs (miRNAs) are short noncoding RNA molecules regulating the expression of mRNAs. Target identification of miRNAs is computationally difficult due to the relatively low homology between miRNAs and their targets. We provide data here utilizing an experimental approach to identify targets of mmu-miR-378-3p, where mmu-miR-378-3p was overexpressed and silenced in NIH-3T3 murine fibroblasts and compared to control RNA transfected cells (RISC-free siRNA). Expression of mRNAs was profiled and differentially expressed genes following either treatment as compared to control transfected cells were identified. In this way we identified 491 significantly differentially expressed genes with more than 1.4 fold change in either comparison. One of the putative targets Akt-1 was subsequently confirmed by luciferase reporter assay.

ORGANISM(S): Mus musculus

PROVIDER: GSE34873 | GEO | 2012/08/08

SECONDARY ACCESSION(S): PRJNA150103

REPOSITORIES: GEO

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Project description:We investigated functions of miRNAs at the level of the whole transcriptome of primary neurons. We transfected mouse E17.5 forebrain primary neuronal cultures (at four to six days of in vitro development) with miRNA mimics and inhibitors. After approximately 48 h post transfection we profiled the effect of these transfections on gene expression with Illumina mRNA microarrays. Cultures were transfected with mimics and inhibitors of five mouse miRNAs (mmu-miR-124, mmu-miR-434-3p, mmu-miR-143, mmu-miR-145 and mmu-miR-25) and with a mimic of a non-mouse miRNA (cel-miR-67). Direct widespread inhibition of gene expression by the perturbed miRNAs was evident when gene expression in cultures transfected with miRNA mimics was compared to those transfected with the inhibitors (or to matched mock transfected cultures): 3-prime UTRs of downregulated transcripts were significantly enriched in seed matching sites for the perturbed miRNAs. Interestingly, analysis of differential gene expression in mock transfected cells (identified through comparison of mock transfected cultures with matched untreated cultures) revealed that genes inhibited by miRNAs were enriched in genes upregulated in mock transfected cultures. This inhibition was the most efficient by the two neuronal miRNAs under investigation (mmu-miR-124 and mmu-miR-434-3p). To investigate if miRNA mediated inhibition of stress induced genes (i.e. stress associated with transfections) was also the case in other stresses, we profiled gene expression changes triggered by chronic neuronal depolarisation. For this, we treated the cultures with KCl (15 mM, 48 h) and compared them to matched untreated cultures. We found that genes upregulated by KCl had a significant intersection with those upregulated by the mock transfection. Moreover, we also found that genes upregulated by KCl had a significant intersection with genes inhibited by mmu-miR-124 and mmu-miR-434-3p. Therefore we concluded that neuronal miRNAs stabilise the neuronal transcriptome by inhibiting stress inducible genes.

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Experimental identification of miR-378-3p targets by overexpression and silencing in murine NIH-3T3 fibroblasts

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