转染三磷酸腺苷酶家族蛋白2 shRNA的人骨肉瘤细胞株U2-OS增殖、凋亡能力观察

Proliferation and apoptosis of human osteosarcoma cell line U2-OS transfected with ATPase family AAA domain-containing protein 2 shRNA

目的观察转染三磷酸腺苷酶家族蛋白2(ATAD2)shRNA的骨肉瘤细胞株U2-OS增殖、凋亡的变化。方法以qRT-PCR法和Western blotting法分别检测人骨肉瘤细胞(U2-OS、MG-63、SaOS-2)和人正常成骨细胞(h FOB1.19)中ATAD2 mRNA、蛋白。在U2-OS细胞中转染ATAD2 shRNA慢病毒载体,采用qRT-PCR法和Western blotting法检测转染效果,CCK8方法检测细胞增殖变化,平板克隆形成实验检测细胞克隆形成数目,碘化丙啶(PI)单染法检测细胞周期,膜联蛋白V-FITC(Annexin V-FITC)/PI双染法检测细胞凋亡率,Western blotting法检测细胞剪切的含半胱氨酸的天冬氨酸蛋白水解酶3(C-Caspase-3)、细胞周期依赖性蛋白激酶4(CDK2)、剪切的含半胱氨酸的天冬氨酸蛋白水解酶9(C-Caspase-9)、细胞周期蛋白D1(CyclinD1)蛋白。结果骨肉瘤细胞中ATAD2 mRNA、蛋白表达水平高于正常成骨细胞(P均<0.05)。ATAD2 shRNA慢病毒载体转染后的骨肉瘤细胞中ATAD2表达水平下降(P<0.05)。转染ATAD2 shRNA后的骨肉瘤细胞增殖能力和克隆形成能力均降低,细胞G1期比例升高,细胞凋亡率升高,细胞中C-Caspase-3、C-Caspase-9蛋白表达升高,CDK2、CyclinD1蛋白表达降低(P均<0.05)。结论转染ATAD2 shRNA可抑制U2-OS细胞增殖,并诱导细胞凋亡。

Objective To observe the changes of proliferation and apoptosis of osteosarcoma cell line U2-OS transfected with ATPase family AAA domain-containing protein 2(ATAD2)shRNA.Methods The qRT-PCR and Western blotting were used to detect ATAD2 mRNA and protein in human osteosarcoma cells(U2-OS,MG-63,and SaOS-2)and human normal osteoblasts(h FOB1.19),respectively.ATAD2 shRNA lentiviral vector was transfected into U2-OS cells,and the effects of transfection were detected by qRT-PCR and Western blotting.Cell proliferation was detected by the CCK8.The number of cell clones was measured by plate clone formation experiments.Propidium iodide(PI)single staining method was used to detect cell cycle,the annexin V-FITC(Annexin V-FITC)/PI double staining to detect apoptosis rate,and Western blotting to detect the cell Cleaved-Caspase-3(C-Caspase-3),cyclin-dependent kinase 2(CDK2),Cleaved-Caspase-9(C-caspase-9)and CyclinD1 proteins.Results The expression levels of ATAD2 mRNA and protein in osteosarcoma cells were higher than those in the normal osteoblasts(all P<0.05).The expression level of ATAD2 in the osteosarcoma cells transfected with ATAD2 shRNA lentiviral vector decreased(P<0.05).After transfection with ATAD2 shRNA,the osteosarcoma cell proliferation ability and colony formation ability were reduced,the proportion of cells in G1 phase increased,the apoptosis rate increased,the levels of C-caspase-3 and C-caspase-9 proteins increased,and the levels of CDK2 and CyclinD1 proteins decreased(all P<0.05).Conclusion Transfection of ATAD2 shRNA could inhibit U2-OS cell proliferation and induce apoptosis.