抑癌基因Runx3增强顺铂致肝癌细胞的凋亡效应

Suppressor Gene Runx3 Enhancing the Cisplatin Induced Apoptosis of Hepatocellular Carcinoma Cells

目的:探讨腺病毒介导的Runx3基因(Ad-Runx3)与化疗药物顺铂(DDP)联合应用对肝癌细胞HLE增殖的抑制效果。方法:检测肝癌细胞HLE中Runx3基因启动子区域甲基化状态及表达情况。通过Ad-Runx3感染肝癌细胞HLE,利用RT-PCR方法检测Runx3基因在肝癌细胞中的转录,用Ad-Runx3联合化疗药物顺铂处理培养的肝癌细胞HLE,利用Western-blot法检测RUNX3在肝癌细胞中高效表达,MTT法检测细胞增殖抑制率,流式细胞术检测细胞周期和凋亡率。结果:在肝癌细胞HLE中Runx3基因启动子区域存在甲基化异常,RT-PCR结果证实HLE中Runx3基因不表达;Ad-Runx3感染HLE细胞后,RT-PCR结果显示有目的基因的转录,并在感染48 h后,RUNX3高效表达。MTT结果显示,100 MOI Ad-Runx3与6.25 mg/L DDP联合应用后5 d,HLE细胞增殖抑制率达(92.46±1.13)%,显著高于单用Ad-Runx3组的(59.28±1.37)%和DDP组的(46.37±2.51)%(均P<0.05)。流式细胞术结果显示,Ad-Runx3与DDP联合应用明显导致细胞S期减少,G2/M期阻滞;Ad-Runx3联合DDP组细胞凋亡率为(18.62±2.48)%,显著高于单用Ad-Runx3组的(8.66±0.78)%和DDP组的(7.48±0.32)%(均P<0.05).结论:Ad-Runx3与DDP联合应用能显著提高对肝癌细胞HLE增殖的抑制作用。

Objective： To investigate the inhibitory effect of adenovirus-mediated Runx3（Ad-Runx3） combined with cisplatin（DDP） on the growth of human hepatocellular carcinoma cell line HLE.Methods： The methylation status of Runx3 was examined by methylation-specific polymerase chain reaction（MSP）,and the expression was detected by RT-PCR.The Runx3 gene was transfected into human hepatocellular carcinoma cell line HLE with a replication-incompetent adenovirus vector,the mRNA transcription of Runx3 gene in HLE cell line was confirmed using RT-PCR method.HLE cells were treated with Ad-Runx3 combined with DDP,and RUNX3 protein expression was detected with Western blot assay.The growth inhibition rate of cells was analyzed by MTT assay,the cell cycle and apoptosis were detected by flow cytometry.Results： HLE cells showed hypermethylation in the promoter region of Runx3 gene by MSP method and inactivation detected by RT-PCR.Runx3 was proved to be successfully transcribed in HLE cells,and efficient RUNX3 protein expression was demonstrated after 48 hours of Ad-Runx3 infection.Five days after combined treatment with Ad-Runx3（100 MOI） and DDP（6.25 mg/L）,the growth inhibitory rate was（92.46±1.13）%,which was significantly higher than（59.28±1.37）% in Ad-Runx3 group and（46.37±2.51）% in DDP group（both P0.05）.Flow cytometry displayed that combined administration of Ad-Runx3 and DDP remarkably reduced S phase and arrested HLE cells at G2/M phase.The cell apoptotic rate was（18.62±2.48）% in Ad-Runx3 and DDP group,which was significantly higher than（8.66±0.78）% in Ad-Runx3 group（8.66±0.78）% and（7.48±0.32）% in DDP group（both P0.05）.Conclusion： Ad-Runx3 combined with DDP has enhanced inhibitory effect on growth of human hepatocellular carcinoma cell line HLE.