Abstract
This study aims to understand the possible effects of TET2 (ten-eleven translocation 2) on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (ADSCs). The human ADSCs were transfected with TET2 siRNAs. The osteogenesis-related genes were detected by quantitative real-time reverse transcription PCR (qRT-PCR), and the osteogenic differentiation was evaluated by alkaline phosphatase (ALP) staining and Alizarin Red staining. TET2 and 5-hydroxymethylcytosine (5hmC) expressions were determined by western blotting and immunofluorescence staining. Meanwhile, wild-type (WT) and TET2-deficient (TET2-/-) mice were selected to observe the alteration of biological characteristics in vivo. TET2 was significantly upregulated along with the osteogenic differentiation of human ADSCs. Compared with Blank group, TET2 siRNA-3 group showed apparent reductions in TET2, 5hmC, and osteogenesis-related genes, as well as decreases in mineralized nodules, ALP activity, and cell growth (all p < 0.05). Besides, Tet2-/- mice had shorter femoral length, lower bone mineral density, and reduced bone volume to total volume (BV/TV) ratio relevant to WT mice; and meanwhile, the percentage of TUNEL-positive chondrocytes increased significantly with the decreased total collagen-positive area, and the distance between two markers of calcein narrowed with declined bone formation rate (BFR) and mineral apposition rate (all p < 0.05). Furthermore, Toluidine Blue staining presented the appreciable decrease of BFR/bone surface (BS) ratio, BFR/BV ratio, osteoblast number over bone perimeter (N.Oc/B.Pm), and osteoblast surface (Ob.S)/BS in Tet2-/- mice (all p < 0.05). Taken together, TET2 upregulation was observed during the osteogenic differentiation of ADSCs, whereas TET2 inhibition may lead to reductions of osteogenesis-related genes and downexpression of 5hmC, which eventually plays a negative role in osteoporosis.
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