High throughput identification of miR-145 targets in human articular chondrocytes

Objective: MicroRNAs play a key role in biological processes, including cartilage development and homeostasis and are dysregulated in many diseases, including osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs. This information is critical if miR-145 is to be considered a target for cartilage repair. Methods: RIP-seq (RNA-immunoprecipitation plus HT-sequencing) of miRISC (miRNA-induced silencing complex) was performed in HACs overexpressing miR-145 to identify miR-145 direct targets. The motif discovery method cWords was used to assess enrichment on miR-145 seed matches in the identified targets. RT-qPCR, Western (immuno-)blot and luciferase assays were used to validate miRNA-target interactions. Results: MiR-145 overexpression affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 39UTR or, more commonly, the coding region. We also demonstrate that miR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion using specific siRNAs lead to MMP13 up-regulation, suggesting that miR-145-mediated DUSP6 depletion contributes to the effect of miR-145 on MMP13 expression. Conclusion: We demonstrate that miR-145 directly targets several genes in primary chondrocytes, including those involved in the expression of the extracellular matrix and inflammation. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.