氯化两面针碱对肝癌细胞SMMC-7721 POLD1基因甲基化及细胞增殖影响研究

Mechanism and effect of Nitidine chloride on the POLD1 gene methylation and the cell proliferation of Liver cancer SMMC-7721

目的探讨氯化两面针碱(nitidine chloride,NC)对肝癌SMMC-7721细胞POLD1基因甲基化及细胞增殖的影响。方法不同剂量的NC(0.5、1.0和2.0mg/mL)作用于人肝癌SMMC-7721细胞48h后,采用焦磷酸技术、RT-PCR和CCK8方法分别测定不同剂量的NC对SMMC-7721细胞中POLD1基因甲基化率,mRNA表达及细胞增殖抑制率的影响。结果不同剂量的NC处理肝癌细胞SMMC-7721 48h后,实验组POLD1基因的甲基化水平整体上升,对照组与实验组甲基化率分别为4.26±0.49、4.14±0.75、4.90±0.64和5.67±0.77。采用方差分析,组间两两比较,1.0、2.0mg/mL实验组与对照组相比较,甲基化率有明显的上升,差异有统计学意义,P值分别为0.041、0.001;0.5mg/mL实验组与对照组相比差异无统计学意义,P>0.05。实验组间两两比较,差异有统计学意义,P值<0.05。对照组和实验组的POLD1基因mRNA相对表达量分别为1.02±0.81、0.83±0.19、0.44±0.09和0.49±0.23,呈下降趋势。秩和检验分析,组间表达差异明显;1.0、2.0mg/mL实验组与对照组相比,差异有统计学意义,P<0.001;0.5mg/mL实验组与对照组相比差异无统计学意义,P>0.05。实验组间两两比较,差异有统计学意义,P<0.05。随着NC浓度的增加,细胞增殖抑制率逐渐增大,分别为21.1%、35.2%和92.1%,呈明显的剂量依赖性。结论 NC可能通过调节SMMC-7721细胞POLD1基因的甲基化水平,增强其启动子的甲基化,抑制POLD1基因表达,从而降低DNA聚合酶δ活性,达到抑制肝癌增殖的作用。

OBJECTIVE To investigate the effects of Nitidine chloride （nitidine chloride, NC）on POLD1 gene methylation, and the proliferation of human hepatoma cells SMMC-7721. METHODS SMMC-7721 cell line were treated with different concentrations of NC（0.5,1.0,2.0 mg/L）. Pyrosequencing, RT-PCR and CCK8 （Celt Counting Kit-8） were used to test the methylation, mRNA levels and growth inhibition ratio of POLD1 gene on the SMMC-7721 cells. RESULTS After starved for 48 h, SMMC-7721 cells were treated with NC （0.5,1.0,2.0 mg/L）. In nitidine chloride group POLD1 gene promoter methylation rates were markedly elevated compared with the control group. In control group and experimental group the methylation rates were 4.26±0.49, 4.14±0.75, 4.90±0.64, 5.67±0.77. ANOVA tests of 1.0 mg/mL and 2.0 mg/mL were used in experimental group, P values were 0. 041 and 0. 001 respectively. POLD1 promoting methylation was obviously higher than that of control group. In 0.5 mg/mL experimental group, there was no statistically significant difference. RT-PCR result showed that POLD1 gene mRNA expression was also on the decline. Comparison between experimental groups, P value was less than 0.05, the difference was statistically significant. The mRNA of POLD1 were 1.02±0. 81, 0. 83±0.19, 0.44±0.09, 0. 49±0.23. Wilcoxon method demonstrated compared to control group, the experimental groups （1.0 and 2.0 mg/mL） has statistically significant difference, P〈0.05. In 0. 5 mg/mL experimental group, there was no statistically significant difference. NC significantly inhibited the proliferation of SMMC-7721 cell lines, the cell proliferation inhibition rates were 21.1%, 35.2%, 92.1%, and it was in a dose dependent manner. CONCLUTIONS Nitidine chloride participates in regulating POLD1 gene methylation levels of liver cancer, particularly enhances the POLD1 gene promoter methylation rates and mediates the transcription activity of POLD1 gene, thus inhibits the activity of DNA polymeraseδ and plays a role in inhibiting liver cancer cell proliferation.