靶向SOX4基因的慢病毒感染对骨肉瘤MG63细胞凋亡的影响
Effects of lentiviral infection targeting SOX4 gene on apoptosis in osteosarcoma cells

目的:探讨靶向SOX4基因的慢病毒感染对骨肉瘤细胞凋亡的影响。方法:体外培养人成骨细胞株hFOB1.19和骨肉瘤细胞株MG63,采用qRT-PCR和Western blot检测两种细胞中SOX4 mRNA和蛋白的表达。将MG63细胞分为对照组、阴性组(感染LV-NC-GFP-shRNA)、干扰组(感染LV-SOX4-GFP-shRNA)和干扰+抑制剂组(LV-SOX4-GFP-shRNA感染前加Caspase-3抑制剂Ac-DEVD-CHO),采用qRT-PCR和Western blot检测对照组、阴性组和干扰组MG63细胞SOX4 mRNA和蛋白的表达情况,用于判断慢病毒感染效果; 采用流式细胞仪检测4组细胞凋亡,Western blot检测酶切Caspase-3蛋白的表达,Caspase-3活性检测试剂盒检测Caspase-3活性,采用JC-1染色检测线粒体膜电位。结果:与hFOB1.19细胞相比,MG63细胞中SOX4 mRNA和蛋白的表达水平升高(P<0.05); 与对照组相比,干扰组MG63细胞中SOX4 mRNA和蛋白的表达降低。干扰组较对照组细胞凋亡增加、Caspase-3活性和酶切蛋白的表达增强,线粒体膜电位降低(P均<0.05),而干扰+抑制剂组上述变化减弱(P<0.05)。结论:SOX4促进MG63细胞凋亡的作用机制可能与Caspase-3介导的凋亡途径有关。
Aim:To explore the effects of lentivirus infection targeting SOX4 gene on osteosarcoma cell apoptosis.Methods:Human osteoblast cell line hFOB1.19 and osteosarcoma cell line MG63 were cultured in vitro.The expressions of SOX4 mRNA and protein were detected by qRT-PCR and Western blot.The MG63 cells were divided into control group(not treated), negative group(negative control LV-NC-GFP-shRNA plasmid infection), interference group(LV-SOX4-GFP-shRNA plasmid infection)and interference+inhibitor group(Caspase-3 inhibitor Ac-DEVD-CHO treatment was added before LV-SOX4-GFP-shRNA plasmid infection). The expressions of SOX4 mRNA and protein in the first 3 groups were detected by qRT-PCR and Western blot to assess the effect of lentivirus infection.The rate of cell apoptosis was detected by flow cytometry,the expression of Caspase-3 protein was tested by Western blot,Caspase-3 activity was measured by Caspase-3 activity assay kit,and the changes of mitochondrial membrane potential were detected by JC-1 staining.Results:The expression levels of SOX4 mRNA and protein in MG63 cells were significantly higher than those of hFOB1.19 cells(P<0.05). Compared with control group,the expressions of SOX4 mRNA and protein in the interference group were reduced(P<0.05).The apoptosis rate of the cells in the interference group was increased,the expression levels of Caspase-3 activity and protein were enhanced,while MTP was decreased(P<0.05)compared with control group; after the addition of Caspase-3 inhibitor, the above changes were significantly weakened(P<0.05).Conclusion:Lentivirus infection targeting SOX4 gene can promote the apoptosis of MG63 cells, and its mechanism may be related to Caspase-3 mediated apoptosis pathway