miRNA-transcriptome profiling of growth plate cartilage

Cartilage originates from mesenchymal cell condensations that differentiate into chondrocytes of transient growth plate cartilage or permanent cartilage of the articular joint surface and trachea. MicroRNAs fine-tune the activation of entire signaling networks and thereby modulate complex cellular responses, but so far only limited data are available on miRNAs that regulate cartilage development. Here we characterize an miRNA which promotes the biosynthesis of a key component in the RAF/MEK/ERK pathway in cartilage. Specifically, by transcriptome profiling we identified miR-322 to be upregulated during chondrocyte differentiation. Among the various miR-322 target genes in the RAF/MEK/ERK pathway, only Mek1 was identified as a regulated target in chondrocytes. Surprisingly, an increased concentration of miR-322 stabilizes Mek1-mRNA to arise protein levels and dampen ERK1/2 phosphorylation, while cartilage-specific inactivation in mice linked the loss of miR-322 to decreased MEK1 levels and increased RAF/MEK/ERK pathway activation. Such mice died perinatally due to tracheal growth restriction and respiratory failure. Hence, a single miRNA can stimulate the production of an inhibitory component of a central signaling pathway to impair cartilage development. Total RNA from microdissected material of the epiphyseal growth plate cartilage of 12-day-old C57BL/6 mice was extracted with phenol-chloroform using standard methods and the RNA integrity was determined (2100 Bioanalyzer, Agilent) according to the manufacturer’s specifications. The material from five epiphyseal growth plates was pooled and the Agilent protocol was applied to label and hybridize 100ng of total RNA per single microarray of a mouse 8x15K-miRBaseV14 slide. After scanning and extraction, data was analyzed using GeneSpring XII software (Agilent). Due to the limited amount of starting material, only two independent hybridization experiments could be performed.