Transfection of primary neuronal cultures with miRNA mimics and inhibitors

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Sergei Manakov 

PROVIDER: E-MTAB-686 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress