Abstract
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.
Highlights
Multipotent mesenchymal stromal cells or stem cells (MSCs) represent a population of adult stem cells that has a potential interest for skeletal tissue engineering, owing to their capacity to differentiate into bone, ligament, tendon or cartilage
The objective of the present study was to identify miRNAs that are regulated during the chondrogenic differentiation of MSCs and eliminating those induced in response to 3D environment or aggregate-induced hypoxia
Expression of miRNAs in MSCs at each time point was normalized to their expression at day 0 and data were presented as the ratio of fold changes in MSCs cultured with TGF-b3 on MSCs cultured without TGF-b3
Summary
Multipotent mesenchymal stromal cells or stem cells (MSCs) represent a population of adult stem cells that has a potential interest for skeletal tissue engineering, owing to their capacity to differentiate into bone, ligament, tendon or cartilage. During the embryonic process of limb formation, MSC condensation is the first step of the differentiation program toward chondrocytes that leads to the formation of cartilage tissue and bone. This differentiation process involves complex signalling pathways that are timely and spacely regulated by several secreted factors [1,2]. Many of these factors are secreted by surrounding tissues and activate signaling cascades leading to proliferation and differentiation of MSCs. Among important genes, the master transcription factor Sox is one of the earliest markers expressed by MSCs undergoing condensation and required for the expression of cartilage-specific matrix proteins [3]. Besides the role of soluble mediators and transcription factors, growing evidence points to the role of epigenetic and microRNA (miRNA)-mediated gene control for initiating and maintaining long-term mature chondrocyte phenotype as well as controlling pathological alterations [4]
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