EphA7在骨肉瘤中的表达及对骨肉瘤MG-63细胞增殖、迁移和凋亡的影响

Expression of EphA7 in osteosarcoma and its effect on proliferation, migration and apoptosis of osteosarcoma MG-63 cells

背景与目的:EphA7蛋白是对人体正常生理过程具有重要作用的一种膜蛋白,常在细胞膜中发挥多种调控作用,但该蛋白在骨肉瘤中的作用尚不清楚。该研究拟观察骨肉瘤组织中EphA7的表达,并了解EphA7对人骨肉瘤MG-63细胞增殖、迁移和凋亡的影响。方法:收集45例患者的骨肉瘤组织和瘤旁正常组织。培养人骨肉瘤MG-63细胞,用siRNA转染MG-63细胞,并分成siRNA-EphA7组(EphA7-siRNA转染)、siRNA-Control组(阴性对照siRNA转染)和空白组(未转染)。采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)和蛋白质印迹法(Western blot)评估组织和细胞中EphA7的mRNA表达和蛋白水平。通过细胞计数试剂盒(cell counting kit-8,CCK-8)测定细胞增殖活力,通过细胞划痕实验检测细胞迁移能力,利用流式细胞术检测细胞凋亡。结果:RTFQ-PCR和Western blot检测结果显示,EphA7的mRNA表达和蛋白水平在骨肉瘤组织中明显高于瘤旁正常组织(P<0.05);与空白组和siRNA-Control组相比,siRNA-EphA7组中EphA7的mRNA表达和蛋白水平显著降低(P<0.05)。CCK-8测定结果显示,siRNA-EphA7组的吸光度(D)值显著低于空白组和siRNA-Control组(P<0.05)。细胞划痕实验显示,与空白组和siRNAControl组相比,siRNA-EphA7组在划痕后愈合率显著降低(P<0.05)。流式细胞术显示,与空白组和siRNA-Control组相比,siRNA-EphA7组凋亡率显著增加(P<0.05)。结论:EphA7在骨肉瘤组织中表达上调。下调MG-63细胞中EphA7的表达可抑制骨肉瘤细胞增殖、迁移,促进其凋亡。

Background and purpose: EphA7 protein is a membrane protein that plays an important role in the normal physiological processes of the human body, and often plays various regulatory roles in the cell membrane. However, the role of this protein in osteosarcoma remains unclear. Methods: Osteosarcoma and adjacent normal tissues of 45 patients were collected. Human osteosarcoma MG-63 cells were cultured and transfected with siRNA, and then divided into siRNA-EphA7 group(EphA7-siRNA transfection), siRNA-Control group(negative control siRNA transfection) and blank group(untransfected). Real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR) and Western blot were used to assess the mRNA and protein expression of EphA7 in tissues and cells. Cell proliferation viability was determined by cell counting kit-8(CCK-8). Cell migration ability was measured by cell scratch assay. Apoptosis was detected by flow cytometry. Results: RTFQ-PCR and Western blot showed that the mRNA expression and protein levels of EphA7 were significantly higher in osteosarcoma than in adjacent normal tissues(P<0.05).The mRNA expression and protein levels of EphA7 in the siRNA-EphA7 group were significantly lower than those in the blank group and the siRNA-Control group(P<0.05). CCK-8 assay showed that the D value of the siRNA-EphA7 group was significantly lower than those of the blank group and the siRNA-Control group(P<0.05). The cell scratch assay showed that the healing rate of the si RNA-Eph A7 group was significantly reduced after scratching compared with the blank group and the siRNA-Control group(P<0.05).Flow cytometry showed a significant increase in apoptotic rate in the siRNA-EphA7 group compared with the blank group and the siRNA-Control group(P<0.05). Conclusion: EphA7 is up-regulated in osteosarcoma, and down-regulation of EphA7 expression in MG-63 cells can inhibit the proliferation and migration of osteosarcoma cells and promote their apoptosis.