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Skeletal development and cartilage formation require stringent regulation of gene expression for mesenchymal stem cells (MSCs) to progress through stages of differentiation. Since microRNAs (miRNAs) regulate biological processes, the objective of the present study was to identify novel miRNAs involved in the modulation of chondrogenesis. We performed miRNA profiling and identify miR-29a as being one of the most down-regulated miRNAs during the chondrogenesis. Using chromatin immunoprecipitation, we showed that SOX9 down-regulates its transcription. Moreover, the over-expression of miR-29a strongly inhibited the expression of chondrocyte-specific markers during in vitro chondrogenic differentiation of MSCs. We identified FOXO3A as a direct target of miR-29a and showed a down- and up-regulation of FOXO3a protein levels after transfection of, respectively, premiR- and antagomiR-29a oligonucleotides. Finally, we showed that using the siRNA or premiR approach, chondrogenic differentiation was inhibited to a similar extent. Together, we demonstrate that the down-regulation of miR-29a, concomitantly with FOXO3A up-regulation, is essential for the differentiation of MSCs into chondrocytes and in vivo cartilage/bone formation. The delivery of miRNAs that modulate MSC chondrogenesis may be applicable for cartilage regeneration and deserves further investigation.

Introduction: Recent evidence suggests that tissue accumulation of senescent p16INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16INK4a in the OA-induced SASP and its regulation by microRNAs (miRs)., Methods: We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis., Results: p16INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16INK4a. Accordingly, p16INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis., Conclusions: We disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.

The aim of this study was to identify new microRNAs (miRNAs) that are modulated during the differentiation of mesenchymal stem cells (MSCs) toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0) and at early time points (day 0.5 and 3) after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXR)α is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.