Pcdna3.1 sirt6

Chemicals and materials MC3T3-E1 obtained from Chinese Academy of Sciences (Shanghai, China). The miR-545-3p mimics and miR-545-3p inhibitor were purchased from Genepharma (Shanghai, China), pcDNA3.1-SIRT6 was constructed by Genepharma (Shanghai, China), Lipofectamine 3000 transfection reagent was obtained from Genepharma (Shanghai, China). Apoptosis assay kit was obtained from Thermo Fisher (Shanghai, China). Alkaline phosphatase (ALP), Run family transcription factor 2 (Runx2) and osteocalcin (OCN), βactin and SIRT6 antibodies were purchased from Abcam (Cambridge, UK).

The miR-545-3p mimics and miR-545-3p inhibitor were purchased from Genepharma (Shanghai, China). pcDNA3.1-SIRT6 was constructed by Genepharma (Shanghai, China), pcDNA3.1 blank vector was used as negative control (NC). MC3T3-E1 were seeded in 6-well plates at the rate of 5 × 10 5 /well. When the cells reached 70% fusion, the cells were transfected by Lipofectamine 3000 (Invitrogen company, USA) with miR-545-3p mimics or miR-545-3p inhibitor or the corresponding negative control (transfected with empty plasmid). The culture medium was changed after 6 hours, and the cells were harvested for realtime qPCR detection after 48 hours, and western blotting was used after 72 hours.