Abstract
BackgroundImproved chondrogenic differentiation of mesenchymal stem cells (MSCs) by genetic regulation is a potential method for regenerating articular cartilage. MiR-127-5p has been reported to promote cartilage differentiation of rat bone marrow MSCs (rMSCs); however, the regulatory mechanisms underlying hypoxia-stimulated chondrogenic differentiation remain unknown.MethodsrMSCs were induced to undergo chondrogenic differentiation under normoxic or hypoxic conditions. Expression of lncRNA DNM3OS, miR-127-5p, and GREM2 was detected by quantitative real-time PCR. Proteoglycans were detected by Alcian blue staining. Western blot assays were performed to examine the relative levels of GREM2 and chondrogenic differentiation related proteins. Luciferase reporter assays were performed to assess the association among DNM3OS, miR-127-5p, and GREM2.ResultsMiR-127-5p levels were upregulated, while DNM3OS and GREM2 levels were downregulated in rMSCs induced to undergo chondrogenic differentiation, and those changes were attenuated by hypoxic conditions (1% O2). Further in vitro experiments revealed that downregulation of miR-127-5p reduced the production of proteoglycans and expression of chondrogenic differentiation markers (COL1A1, COL2A1, SOX9, and ACAN) and osteo/chondrogenic markers (BMP-2, p-SMAD1/2). MiR-127-5p overexpression produced the opposite results in rMSCs induced to undergo chondrogenic differentiation under hypoxic conditions. GREM2 was found to be a direct target of miR-127-5p, which was suppressed in rMSCs undergoing chondrogenic differentiation. Moreover, DNM3OS could directly bind to miR-127-5p and inhibit chondrogenic differentiation of rMSCs via regulating GREM2.ConclusionsOur study revealed a novel molecular pathway (DNM3OS/miR-127-5p/GREM2) that may be involved in hypoxic chondrogenic differentiation.
Highlights
Improved chondrogenic differentiation of mesenchymal stem cells (MSCs) by genetic regulation is a potential method for regenerating articular cartilage
We analyzed the levels of chondrogenic differentiation protein markers (COL1A1, COL2A1, SOX9, and ACAN) in normoxic or hypoxic conditions (1% O 2)
We detected the levels of miR-127-5p at 7 and 14 days in rat bone marrow MSCs (rMSCs) by RT-qPCR
Summary
Improved chondrogenic differentiation of mesenchymal stem cells (MSCs) by genetic regulation is a potential method for regenerating articular cartilage. Mesenchymal stem cells (MSCs) are a kind of cells with the ability of multi-differentiation, and could possibly be used for cell-based regenerative therapy of articular cartilage defects [9]. Persistent exposure of MSCs to hypoxia can downregulate their levels of CBFA-1/Runx, osteocalcin, and type I collagen [16] These findings regarding the role played by hypoxia in MSC differentiation remain controversial because the relevant studies used different hypoxia exposure times and sources of MSCs. These findings regarding the role played by hypoxia in MSC differentiation remain controversial because the relevant studies used different hypoxia exposure times and sources of MSCs Those studies prompted us to explore the mechanisms that regulate the differentiation of MSCs into chondrocytes under hypoxic conditions
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