Introduction: Axonal growth is essential for the establishment of a functional neuronal network. Molecular information of axon is limited. MicroRNAs (miRNAs) regulate post-transcriptional gene expression. We hypothesized that axonal miRNAs are locally relevant to their target genes.
Methods: Proteins and RNAs were extracted from distal axons of cortical neurons cultured in a microfluidic device. A mass spectrometer and miRNA arrays were used to measure proteins and miRNAs, respectively. Ingenuity Pathway Analysis (IPA) and Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tools were used to make in silico predictions of functionally relevant miRNA target genes.
Results: Proteomic showed that distal axons contained 883 proteins. Bioinformatic analysis showed the presence of 94 proteins that regulate axonal growth. To identify relevant miRNAs to these 94 proteins, miRNAs with 8mer sites that exactly match target genes were considered, based on the fact that 8mer sites efficaciously affect miRNA-target interactions. Of the 94 genes, we found that there were 56 candidate genes that can be targeted by 62 miRNAs enriched in axons. Among them, we validated 13 proteins and 11 miRNAs, respectively, by means of Western blot and RT-PCR. To examine target genes, we treated axons with chondroitin sulfate proteoglycans (CSPGs) that inhibit axonal growth and examined alterations of these proteins and miRNAs in the distal axons. We found that elevation of miR-203a, -133b, -29abc and -92ab were associated with reduced AKT, MTOR, PI3Kp85, DPYSL2, MAP1B, PPP2CA and DCX proteins, whereas decreased miR-15b, -26b, -34b, -376b, -128, -381 and -195 were accompanied by increased proteins of EZR, KIF5A, RTN4, GSK3B, and ROCK2. Bioinformatic analysis revealed that these miRNAs and proteins are highly related to the axonal growth network. These data suggest that miRNAs altered by CSPGs functionally target these genes for mediating the inhibitory effect of CSPGs on axonal growth.
Conclusions: Our bioinformatic analyses of miRNAs and proteins in the distal axon identifies an interconnected group of miRNAs and their target genes that regulate axonal growth, which provides new insight into the molecular mechanisms underlying axonal growth.