MiR-152 influences osteoporosis through regulation of osteoblast differentiation by targeting RICTOR.

Context: Evidence suggests that microRNA (miRNA) regulate gene expression and bone tissue homoeostasis of osteoporosis. MiR-152 has found to be abnormally expressed in osteoporosis, but its role in osteoblast differentiation has not been elucidated. Objective: To understand the potential mechanism of miR-152 in osteoblast differentiation via regulation of RICTOR. Materials and methods: The expression of miR-152 and RICTOR were tested in ovariectomized rat models of osteoporosis. Primary osteoblasts and MC3T -E1 cells were assigned into four groups, namely Control, miR-152 inhibitor, miR-control and miR-152 inhibitor + siRICTOR groups. qRT PCR and Western blot were performed to detect the expressions of miR-152 and RICTOR, respectively. MTT assay was used to evaluate cell viability, and ALP activity determination and mineralization analyses were also conducted. Results: In ovariectomy-induced osteoporotic rats, miR-152 (3.06 ± 0.35) in femoral tissues increased significantly, while RICTOR (0.31 ± 0.04) decreased. Compared with Control group, miR-152 inhibitor group presented appreciable reduction of miR-152 in primary osteoblasts and MC3T3-E1 cells, as well as remarkable increases in RICTOR, p-Akt(s473)/Akt ratio, and osteogenesis-related genes, with enhanced cell viability, ALP activity and mineralization. In comparison with cells in the miR-152 inhibitor group, those in the miR-152 inhibitor + siRICTOR group had no observable difference in miR-152, but were dramatically up-regulated in RICTOR, as well as the corresponding opposite tendencies of other factors. Conclusion: Inhibiting miR-152 promoted osteoblasts differentiation and alleviated osteoporosis by up-regulating RICTOR. Therefore, miR-152 may be an essential mediator of osteoblast differentiation and a new therapeutic strategy for osteoporosis.

The Positive Effect of TET2 on the Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

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.