MiR-335-5p promotes chondrogenesis in mouse mesenchymal stem cells and is regulated through two positive feedback loops

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is regulated by many factors and signal pathways, including transcription factors such as Sox9 and microRNAs. MiR-335-5p has been previously reported to regulate osteogenic and adipogenic differentiations of MSCs, but its role in chondrogenic differentiation of MSC remains unknown. In this study, we found that miR-335-5p and its host gene Mest are co-expressed and greatly upregulated during mouse MSCs (mMSCs) chondrogenesis. Overexpression of miR-335-5p in mMSCs increased expression of chondrogenic marker genes. Molecular mechanism explorations revealed that miR-335-5p targets Daam1 and ROCK1, a set of negative regulators of Sox9; Sox9 downregulates the expression of miR-29a and 29b, both negative regulators of Mest expression, thus forming a positive loop from miR-335-5p to Sox9 to Mest/miR-335-5p. In addition, miR-335-5p targets DKK1 during mMSC chondrogenic differentiation to increase β-catenin/TCF activity, which leads to increased level of Mest transcription. These data showed miR-335-5p positively regulates MSC chondrogenesis, and two positive feedback loops are identified for the expression of miR-335-5p and its host gene Mest during the early phase of mMSC chondrogenic differentiation.