姜黄素对非小细胞肺癌A459细胞增殖及CDH13甲基化状态的影响

Effect of curcumin on proliferation and CDH13 methylation status of non-small-cell lung cancer cell line A549

目的观察姜黄素对非小细胞肺癌(NSCLC)A549细胞DNA甲基化转移酶(Dnmts)活性和抑癌基因人H-钙黏蛋白(CDH13)的影响,探讨姜黄素的去甲基化机制。方法将A549细胞分为两组,观察组加入40μmol/L姜黄素和培养液,对照组仅加入培养液。采用MTT法检测两组细胞增殖能力,RT-PCR方法检测CDH13 mRNA表达,甲基化特异性PCR(MSP)检测CDH13启动子CpG岛的甲基化状态,酶活性连续循环比色法检测Dnmt1、Dnmt3b活性。结果观察组和对照组的细胞增殖OD值分别为0.638±0.184、0.859±0.040,观察组低于对照组(P<0.05)。两组CDH13 mRNA的相对表达量分别为0.64±0.09、0.43±0.12,观察组高于对照组(P<0.05)。对照组出现甲基化扩增条带,无去甲基条带;观察组除甲基化条带外,出现去甲基化条带。观察组和对照组每mg核蛋白中Dnmt1的活性分别为(0.153±0.016)、(0.770±0.014)μmol/min,Dnmt3b的活性分别为(0.137±0.020)、(0.179±0.013)μmol/min,观察组Dnmt1、Dnmt3b活性均较对照组下降(P均<0.05)。结论姜黄素可通过降低Dnmts活性来抑制CDH13的甲基化状态,从而抑制A549细胞增殖。

Objective To observe the effect of curcumin on DNA methyltransferase（Dnmts） activity and tumor suppressor gene human H-cadherin（CDH13） in non-small-cell lung cancer（NSCLC） A549 cells and to explore the mechanism of curcumin＇s demethylation.Methods A549 cells were divided into two groups：the observation group and the control group.Cells in the observation group were cultured with 40 μmol/L curcumin and the culture medium,while cells in the control group only with the culture medium.MTT assay was used to detect the cell proliferation,and RT-PCR was used to detect the expression of CDH13 mRNA.Cp G island methylation status of CDH13 promoter was detected by methylationspecific PCR（MSP）,and catalytic activity of Dnmts was detected by enzyme colorimetric determination kit.Results The OD of the observation group was 0.638±0.184,which was lower than that（0.859±0.040） of the control group（P〈0.05）.The relative expression level of CDH13 mRNA in the observation group was 0.64±0.09,which was higher than that（0.43±0.12） of the control group.There were methylation amplification bands,but no demethylation bands in the control group,and there were both methylation amplification bands and demethylation bands in the observation group.In the observation group and the control group,the activities of Dnmt1 were separately（0.153±0.016） and（0.770±0.014） μmol/min,and the activities of Dnmt3 b were（0.137±0.020） and（0.179±0.013） μmol/min,respectively.The activities of Dnmt1 and Dnmt3 b in the observation group were lower than those in the control group（all P〈0.05）.Conclusion Curcumin inhibits the methylation status of CDH13 by decreasing the activity of Dnmts,and thereby inhibits A549 cell proliferation.