BACKGROUND: Chondrogenic differentiation of mesenchymal stem cells (MSCs) is important for osteoarthritis (OA) treatment. However, the specific mechanisms involved are undefined. MicroRNAs (miRNAs) downregulate protein synthesis by binding to the 3'UTR of target mRNA. METHODS: Bone marrow aspirates were obtained from OA patients undergoing total hip arthroplasty (n=8) to isolate MSCs. MiR-410 or miR-410 inhibitor were transfected into MSCs using lentivirus and the effects were assessed. Alcian blue staining detected differences in chondrogenic differentiation. An MTT assay and flow cytometry determined changes in cell proliferation and cell cycle, respectively. Real time PCR assessed differences in miRNA and mRNA expression levels and western blotting detected changes in protein levels. ChIP assessed differences in transcriptional activation. TOP/FOP determined changes in the activity of the Wnt signaling pathway. A dual-luciferase reporter assay was used to confirm the miR-410 target protein. RESULTS: miR-410 was elevated during transforming growth factor β3 (TGF-β3)-induced chondrogenic differentiation of MSCs. miR-410 targeted a putative binding site in the 3'-UTR of the Wnt3a gene, thus regulating the Wnt signaling pathway. miR-410 transfection increased mRNA and protein levels of four chondrogenic markers, type II collagen (Col2a1), SRY-box 9 (Sox9), aggrecan (ACAN), and hyaluronan synthase 2 (Has2). miR-410 overexpression decreased Wnt3a protein expression. Wnt3a levels increased in OA patient cartilage concomitant with OA severity and significantly negatively correlated with miR-410 levels. CONCLUSION: miR-410 is a key regulator of MSC chondrogenic differentiation and directly targets Wnt3a triggering the Wnt signaling pathway.
BACKGROUND: Chondrogenic differentiation of mesenchymal stem cells (MSCs) is important for osteoarthritis (OA) treatment. However, the specific mechanisms involved are undefined. MicroRNAs (miRNAs) downregulate protein synthesis by binding to the 3'UTR of target mRNA. METHODS: Bone marrow aspirates were obtained from OA patients undergoing total hip arthroplasty (n=8) to isolate MSCs. MiR-410 or miR-410 inhibitor were transfected into MSCs using lentivirus and the effects were assessed. Alcian blue staining detected differences in chondrogenic differentiation. An MTT assay and flow cytometry determined changes in cell proliferation and cell cycle, respectively. Real time PCR assessed differences in miRNA and mRNA expression levels and western blotting detected changes in protein levels. ChIP assessed differences in transcriptional activation. TOP/FOP determined changes in the activity of the Wnt signaling pathway. A dual-luciferase reporter assay was used to confirm the miR-410 target protein. RESULTS:miR-410 was elevated during transforming growth factor β3 (TGF-β3)-induced chondrogenic differentiation of MSCs. miR-410 targeted a putative binding site in the 3'-UTR of the Wnt3a gene, thus regulating the Wnt signaling pathway. miR-410 transfection increased mRNA and protein levels of four chondrogenic markers, type II collagen (Col2a1), SRY-box 9 (Sox9), aggrecan (ACAN), and hyaluronan synthase 2 (Has2). miR-410 overexpression decreased Wnt3a protein expression. Wnt3a levels increased in OA patientcartilage concomitant with OA severity and significantly negatively correlated with miR-410 levels. CONCLUSION:miR-410 is a key regulator of MSC chondrogenic differentiation and directly targets Wnt3a triggering the Wnt signaling pathway.