过表达SIRT4对骨肉瘤细胞恶性生物学行为及能量代谢的影响

Effect of Sirtuin 4 overexpression on malignant biological behavior and energy metabolism of osteosarcoma cells

目的:探讨过表达SIRT4对人骨肉瘤细胞恶性生物学行为及对成骨分化的调控作用,以及过表达SIRT4对线粒体能量代谢的影响。方法:构建稳定过表达SIRT4的U2OS、MG63骨肉瘤细胞株,通过qRT-PCR和Western blot检测SIRT4的mRNA和蛋白表达水平;CCK-8、克隆形成实验、EDU555染色法及检测细胞增殖;裸鼠胫骨成瘤实验检验成瘤能力;流式细胞术检测细胞周期;DAPI染色及Annexin V-APC/7-AAD双染法检测细胞凋亡;细胞划痕实验、Transwell实验检测细胞迁移及侵袭能力;碱性磷酸酶(alkaline phosphatase,ALP)染色和茜素红S染色分别检测细胞的早期和晚期成骨分化;葡萄糖检测试剂盒测定培养基及细胞内葡萄糖含量;乳酸检测试剂盒测定培养基及细胞内乳酸含量;三磷酸腺苷(adenosine triphosphate,ATP)检测试剂盒检测细胞总ATP含量;四甲基罗丹明酯染色(tetramethylrhodamine ethylester perchlorate,TMRE)测定试剂检测线粒体膜电位。结果:过表达SIRT4组细胞增殖、迁移及侵袭能力降低,裸鼠胫骨成瘤的瘤体体积减小,凋亡细胞增多;过表达SIRT4组细胞的G_(0)/G_(1)期占比明显增多,S期占比明显减少;过表达SIRT4组细胞ALP染色、茜素红S染色钙盐结节明显增多;过表达SIRT4组培养基中的葡萄糖较对照组消耗减少,细胞中的葡萄糖含量较对照组减少;过表达SIRT4组细胞中的乳酸含量较对照组减少,培养基中的乳酸较对照组产生减少;过表达SIRT4组细胞的总ATP较对照组更低;过表达SIRT4组的线粒体膜电位较对照组更低。结论:在U2O和MG63细胞中,过表达SIRT4抑制骨肉瘤细胞增殖、迁移、侵袭以及成瘤能力,促进骨肉瘤细胞凋亡以及早晚期成骨分化,调节骨肉瘤细胞能量代谢。

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 metabolism.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 ability;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 observe the migration and invasion abilities of cells;alkaline phosphatase(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 reductions 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 increase in the proportion of cells in G_(0)/G_(1) phase and a significant reduction in the proportion of cells in S phase.ALP staining and alizarin 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 overexpression group had a lower mitochondrial membrane potential than the control group.Conclusion:In U2O and MG63 cells,SIRT4 overexpression inhibits the proliferation,migration,invasion,and tumorigenic ability of osteosarcoma cells,promotes the apoptosis of osteosarcoma cells and early and late osteogenic differentiation,and regulates energy metabolism in osteosarcoma cells.