In vitro: Cells were seeded in a 96-well plate at the density of 1.5 × 104 per well. The cells were transfected with Ndrg1 expression construct or the vector for 4 h in presence of the JetPRIME transfection reagent. The effect of NDRG1 on cell growth was determined by using a CCK-8 cell counting kit. In vivo transfection of Ndrg1 siRNA was done and its effect on osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells in mice was explored: C57BL/6 mice were purchased from SPF Biotechnology and housed in SPF facility with a 12 h:12 h light/dark cycle and free access to food and water. The in vivo efficacy of the Ndrg1 siRNA was tested in mice 3 days after receiving intra-tibial transfection of 10 μg 2’-Ome modified Ndrg1 siRNA or control siRNA (Genepharma, Shanghai, China). The in vivo transfection procedure was described previously. Briefly, for one mouse, 10 μg 2’-Ome modified siRNA and 1.5 μL in vivo-jetPEI (Polyplus, Illkirch, France), each diluted with 12.5 μl of 5% glucose, were mixed by vortexing. Then, the complexed siRNA was delivered to the marrow of the mouse tibiae. BMSCs were flushed from the tibiae, grown in α-MEM containing 10% FBS, subjected to RNA extraction and thereafter qRT-PCR. To examine the in vivo role of NDRG1, the female mice aged 8 weeks were randomly allocated into four groups: Sham/Ctrl siRNA group (sham-operated mice receiving control siRNA), OVX/Ctrl siRNA group (ovariectomized mice receiving control siRNA), Sham/Ndrg1 siRNA group (sham-operated mice receiving Ndrg1 siRNA) and OVX/Ndrg1 siRNA group (ovariectomized mice receiving Ndrg1 siRNA). The mice received ovariectomy or sham surgery 7 days after the first transfection, and received additional transfections every 3 weeks after the first transfection. 4 weeks after the surgery, some mice were sacrificed and BMSCs were isolated from the transfected tibiae and cultured in α-MEM containing 10% FBS. The cells at passage 3 were subjected to protein extraction and Western blotting analysis of Wnt/β-catenin pathway proteins. Additionally, the cells at passage 3 were induced to allow osteogenic or adipogenic differentiation. The other mice were sacrificed 12 weeks after the first transfection and the tibiae were collected.