Effect of Sirtuin 4 overexpression on malignant biological behavior and energy metabolism of osteosarcoma cells
Objective:To investigate the regulatory effect of sirtuin 4(SIRT4)overexpression on the malignant biological behavior of human osteosarcoma cells and osteogenic differentiation,as well as the influence of SIRT4 overexpression on mitochondrial energy me-tabolism.Methods:U2OS and MG63 osteosarcoma cell lines with stable overexpression of SIRT4 were constructed,and qRT-PCR and Western blot were used to measure the mRNA and protein expression levels of SIRT4;CCK-8 assay,colony formation assay,and EDU555 staining were used to observe cell proliferation;tibial tumorigenesis assay in nude mice was used to observe tumorigenic abil-ity;flow cytometry was used to detect cell cycle;DAPI staining and Annexin V-APC/7-AAd double staining were used to measure cell apoptosis;wound healing assay and Transwell assay were used to ob-serve the migration and invasion abilities of cells;alkaline phospha-tase(ALP)staining and alizarin red S staining were used to detect early and late osteogenic differentiation,respectively;glucose assay kit was used to determine the content of glucose in the medium and cells;lactic acid assay kit was used to determine the content of lactic acid in the medium and cells;triphosphate assay kit was used to determine the total content of adenosine triphosphate(ATP)in the medium and cells;tetramethylrhodamine ethyl ester perchlorate staining was used to measure mitochondrial membrane potential.Results:The SIRT4 overexpression group had significant reduc-tions in the proliferation,migration,and invasion abilities of cells,a significant reduction in the tumor volume of tibial osteoblastoma in nude mice,and a significant increase in apoptotic cells.As for cell cycle,the SIRT4 overexpression group showed a significant in-crease in the proportion of cells in G0/G1 phase and a significant reduction in the proportion of cells in S phase.ALP staining and aliza-rin red S staining showed a significant increase in the number of calcium salt nodules in the SIRT4 overexpression group.Compared with the control group,the SIRT4 overexpression group had significant reductions in the consumption of glucose in the medium and the content of glucose in cells,as well as significant reductions in the content of lactic acid in cells and the medium.The SIRT4 overexpres-sion group had a lower mitochondrial membrane potential than the control group.Conclusion:In U2O and MG63 cells,SIRT4 overex-pression inhibits the proliferation,migration,invasion,and tumorigenic ability of osteosarcoma cells,promotes the apoptosis of osteosar-coma cells and early and late osteogenic differentiation,and regulates energy metabolism in osteosarcoma cells.
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