High through-put identification of miR-145 targets in human articular chondrocytes
Life(2020)
摘要
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 3’UTR 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.
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